WO2011072247A2 - Facteurs de restriction pathogènes - Google Patents

Facteurs de restriction pathogènes Download PDF

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WO2011072247A2
WO2011072247A2 PCT/US2010/059934 US2010059934W WO2011072247A2 WO 2011072247 A2 WO2011072247 A2 WO 2011072247A2 US 2010059934 W US2010059934 W US 2010059934W WO 2011072247 A2 WO2011072247 A2 WO 2011072247A2
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cell
infection
gene
virus
cells
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WO2011072247A3 (fr
WO2011072247A8 (fr
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Abe Brass
Steve Elledge
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Brigham and Womens Hospital Inc
General Hospital Corp
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Brigham and Womens Hospital Inc
General Hospital Corp
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Priority to EP10836770A priority patent/EP2510087A2/fr
Publication of WO2011072247A2 publication Critical patent/WO2011072247A2/fr
Publication of WO2011072247A8 publication Critical patent/WO2011072247A8/fr
Publication of WO2011072247A3 publication Critical patent/WO2011072247A3/fr
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Definitions

  • This invention relates to the use of Interferon Induced Trans-membrane Proteins (IFITM proteins) as pathogen restriction factors for numerous viruses and other intracellular pathogens, e.g., to inhibit infection by those pathogens, methods of using the same to produce the pathogens, and transgenic animals expressing exogenous IFITMs, e.g., IFITM 1, 2, and 3.
  • IFITM proteins Interferon Induced Trans-membrane Proteins
  • Influenza epidemics exact a daunting toll on world health. Moreover, viral super-infections can produce antigenic shifting, resulting in more virulent pathogens (Monto, Clin Infect Dis 48 Suppl 1, S20-25, 2009). At present, the emergence of a novel influenza A H IN 1 viral strain has created a pandemic, producing illness in over 70 countries. Additionally, the related avian influenza A viral strain, H5N1, represents a potentially catastrophic global health risk (Maines et al, Clin Infect Dis 48 Suppl 1, S20- 25, 2008).
  • the influenza A viral genome encodes for 11 proteins and consists of eight segments of negative single-stranded RNA (Lamb and Krug, Orthomyxoviridae: The viruses and their replication., 4th edn, Philadelphia, Lippincott Williams and Wilkins, 2001). Each sub-genomic segment is coated by viral nucleoprotein (NP) and bound to a single viral RNA-dependent RNA-polymerase holoenzyme (RdRp), composed of PA, PB 1 and PB2 subunits.
  • NP viral nucleoprotein
  • RdRp RNA-dependent RNA-polymerase holoenzyme
  • HA hemagglutinin
  • sialyated host cell surface glycoproteins Cho and Whittaker, Proc Natl Acad Sci U S A lOl, 18153-18158, 2004; Skehel and Wiley, Am J Respir Crit Care Med 152, S13-15, 1995.
  • viral particles are trafficked through both early and late endosomes, with the intense acidification of the latter compartment altering the conformation of HA, leading to host-viral membrane fusion, and entry of the vRNPs into the cytosol (Sieczkarski and Whittaker, Traffic 4, 333-343, 2003).
  • Nuclear localization signal sequences contained in NP, PB1 and/or PB2 are then bound by host cell karyopherins, and the vRNPs are transported though the nuclear pore complex (NPC, (Boulo et al, Virus Res 124, 12-21, 2007)).
  • the RdRp commandeers 5' caps from host mRNAs to prime transcription of viral mRNA (vmRNA, (Bouloy et al, Proc Natl Acad Sci U S A 75, 4886- 4890, 1978) (Engelhardt and Fodor, Rev Med Virol 16, 329-345, 2006)).
  • vmRNA viral mRNA
  • the RdRp creates a positive sense template (cDNA), from which it synthesizes new viral genomes (vRNAs).
  • cDNA positive sense template
  • the vRNAs are coated by NP and exported though the NPC by the viral factors Ml and NEP/NS2 (nuclear export protein) working in concert with the host nuclear export machinery.
  • the viral envelope proteins HA, M2 and neuraminidase (NA) are translated on the rough endoplasmic reticulum (ER) and trafficked to the cell surface where they, along with the soluble factors Ml, RdRp and eight distinct vRNPs, are packaged into budding virions.
  • IFNs orchestrate a large component of this anti-viral response, at both a cellular and organismal level (Grandvaux et al, Curr Opin Infect Dis 15, 259-267, 2002).
  • gene products are differentially regulated after IFN stimulation, including the important downstream anti-viral effectors MxA, PKR, RIG-I, and 2'5'-OAS (Grandvaux et al, Curr Opin Infect Dis 15, 259-267, 2002; Haller et al, Rev Sci Tech 28, 219-231., 2009; Nakhaei et al, Semin Immunol 21, 215-222, 2009).
  • many viruses deploy anti-IFN countermeasures, which for influenza A virus are primarily enacted by the viral protein, NS1 (Hale et al, J Gen Virol 89, 2359-2376, 2008).
  • the present invention is based, at least in part, on the discovery that IFITMl, 2 and 3 are viral restriction factors, i.e., host cell proteins that inhibit viral replication.
  • the invention provides isolated cells that have been engineered to specifically disrupt or reduce expression of an interferon induced transmembrane protein 1, 2, or 3 (IFITMl, 2, or 3) protein.
  • the cells are more susceptible to infection with a virus, parasite, or bacterium, or to a bacterial toxin, that is endocytosed, than a wild-type cell of the same type having normal expression of the IFITMl, 2, or 3.
  • the cell is infected with a virus, parasite or bacterium.
  • the virus is selected from the group consisting of orthomyxoviruses, flaviviruses, Hepadnaviruses, Hepeviruses, Picornaviridae, and retroviruses.
  • the virus is selected from the group consisting of RNA viruses, and DNA viruses.
  • the bacterium, parasite, or toxin is selected from the group consisting of Gram-negative bacteria; Gram-positive bacteria; fungi; protozoa; and bacterial toxins.
  • the cell is a human cell, such as PER.C6, or HEK293 cell, a non-human mammalian cell (such as African green monkey kidney (Vero or COS cells), Chinese hamster ovary cells (CHO), or Madin-Darby canine kidney (MDCK) cells), a transformed or primary chicken cell, or an avian embryonated egg cell (such as from a chicken).
  • the cells can also be stem cells.
  • the cell is a mammalian cell, e.g., a human cell, and the cell has been engineered to specifically disrupt or reduce expression of one or both of IFITM2 and IFITM3.
  • the cell is a bird cell or a pig cell, and the cell has been engineered to specifically disrupt or reduce expression of IFITMl .
  • the IFITM protein is at least 95% identical to NCBI Reference Sequence: NP_066362.2 interferon-induced transmembrane protein 3 (1-8U) [Homo sapiens] (SEQ ID NO:598), NCBI Reference Sequence: NP_006426.2 interferon induced transmembrane protein 2 (1-8D) [Homo sapiens] (SEQ ID NO:599), or NCBI Reference Sequence: NP_003632.3 interferon induced transmembrane protein 1 (9-27) [Homo sapiens] (SEQ ID NO:600).
  • the invention provides methods for producing a virus, parasite, bacterium, or toxin.
  • the methods include obtaining a host cell that has been engineered to specifically disrupt or reduce expression of a pathogen restriction factor, e.g., a viral restriction factor, e.g., an interferon induced transmembrane protein 1, 2, or 3 (IFITMl, 2, or 3), PULS 1, TPST1, or WDR33, e.g., a host cell as described herein; infecting the host cell with the virus, parasite, or bacterium; maintaining the host cell under conditions sufficient for the virus or bacterium to be produced, and isolating the virus or bacterium produced by the cell.
  • a pathogen restriction factor e.g., a viral restriction factor, e.g., an interferon induced transmembrane protein 1, 2, or 3 (IFITMl, 2, or 3), PULS 1, TPST1, or WDR33, e.g., a host cell as described here
  • the host cell is an isolated host cell, and the host cell is maintained in media, and the virus, parasite, bacterium, or toxin is isolated from the host cell or the media.
  • the pathogen is a virus.
  • the invention provides transgenic animals, the nucleated cells of which comprise a transgene encoding IFITMl, 2, or 3, wherein the animals exhibit a decreased susceptibility to viral infection as compared to a wildtype animal.
  • the animal is a pig, chicken, duck, or turkey.
  • the invention provides methods for treating or reducing risk of a viral or bacterial infection in a subject.
  • the methods include administering to the subject a therapeutically effective amount of a composition comprising an IFITMl, 2, or 3 protein, in a physiologically acceptable carrier that promotes incorporation of the IFITMl, 2, or 3 protein into the membrane of cells of the subject.
  • the composition includes the IFITMl, 2, or 3 protein incorporated into a liposomal preparation.
  • the IFITM protein is at least 95% identical to NCBI Reference Sequence: NP_066362.2 interferon-induced transmembrane protein 3 (1-8U) [Homo sapiens] (SEQ ID NO:598), NCBI Reference Sequence: NP_006426.2 interferon induced transmembrane protein 2 (1-8D) [Homo sapiens] (SEQ ID NO:599), or NCBI Reference Sequence: NP_003632.3 interferon induced transmembrane protein 1 (9-27) [Homo sapiens] (SEQ ID NO:600).
  • the invention features methods for identifying a candidate compound that modulates viral infection.
  • the methods include selecting a target gene from Table 1 or Table 2; providing a sample comprising the target gene, e.g., a cell expressing the target gene; contacting the sample with a test compound; and evaluating expression or activity of the target gene in the presence of the test compound.
  • a test compound that modulates, e.g., increases or decreases, expression or activity of the target gene in the presence of the test compound as compared to expression or activity of the target gene in the absence of the test compound is a candidate compound that modulates viral infection.
  • a test compound that decreases expression of a gene listed in Table 1, or increases expression of a gene listed in Table 2 is a candidate compound for decreasing or inhibiting viral infection
  • a test compound that increases expression of a gene listed in Table 1. or decreases expression of a gene listed in Table 2. is a candidate compound for increasing or promoting viral infection.
  • the methods further include selecting a candidate compound that decreases expression of a gene listed in Table 1, or increases expression of a gene listed in Table 2; providing a cell or animal model of an infection, e.g., a viral infection, e.g., infection with influenza A; and detecting an effect of the candidate compound on infection in the cell or animal model.
  • a candidate compound that decreases or inhibits infection in the cell or animal model is a candidate therapeutic compound for the treatment of the infection.
  • the invention features methods for treating or inhibiting a viral infection in a subject or a cell.
  • the methods include administering to the subject or cell a composition comprising an inhibitor of a gene or protein listed in Table 1.
  • the inhibitor is an siRNA that specifically decreases expression of a gene listed in Table 1 , e.g., an siRNA listed in Table 1.
  • the invention provides animals, e.g., a population of non-human animals, possessing a functionally deleted form of a gene set forth in Table 2, wherein the population is more susceptible to infection by a pathogen.
  • the invention provides animals, e.g., a population of non-human animals possessing a functionally deleted form of a gene set forth in Table 1, wherein the population is less susceptible to infection by a pathogen.
  • the invention features methods for identifying a compound that binds to a gene product set forth in Table 1 or Table 2 and can decrease infection of a cell by a pathogen.
  • the methods include contacting a compound with a gene product set forth in Table 1 or 2; detecting binding of the compound to the gene product; and associating binding with a decrease in infection by the pathogen.
  • the methods also include optimizing a compound that binds the gene product in an assay that determines the functional ability to decrease infection, e.g., a cell based assay or an in vivo assay.
  • the invention provides methods for identifying an agent that decreases infection of a cell by a pathogen.
  • the methods include administering the agent to a cell containing a cellular gene encoding a gene product set forth in Table 1; detecting the level and/or activity of the gene product produced by the cellular gene, a decrease or elimination of the gene product and/or gene product activity indicating an agent with antipathogenic activity.
  • the activity is binding between a gene product set forth in Table 1 and another cellular protein or binding between a gene product set forth in Table 1 and a pathogenic (i.e., non-host) protein.
  • the invention features methods for identifying an agent that decreases infection in a cell by a pathogen. The methods include administering the agent to a cell containing a cellular gene encoding a gene product set forth in Table 1 ; contacting the cell with a pathogen; and determining the level of infection, a decrease or elimination of infection indicating that the agent is an agent that decreases infection.
  • the methods include measuring the level of expression and/or activity of the gene product.
  • the level of infection is determined by determining the level of replication of the pathogen.
  • the pathogen is a virus.
  • the invention features methods for inhibiting infection in a cell by a pathogen comprising decreasing expression or activity of a gene or gene product set forth in Table 1.
  • the infection is decreased by decreasing the replication of the pathogen.
  • the pathogen is a virus.
  • expression or activity of the gene or gene product is decreased by contacting the cell with a composition comprising a chemical, a compound, a small molecule, an aptamer, a drug, a protein, a cDNA, an antibody, a morpholino, a triple helix molecule, an siRNA, LNA, an shRNAs, an antisense nucleic acid or a ribozyme.
  • decreasing expression comprises decreasing translation of an mR A encoding the gene product set forth in Table 1.
  • the composition comprises an antisense nucleic acid that specifically hybridizes and decreases expression or activity of the gene product, e.g., an siRNA that decreases expression or activity of the gene product.
  • the composition comprises an antibody that specifically binds to a protein.
  • FIG. 1A is a set of three images of U20S cells transfected with the indicated siRNAs for 72 hours, then infected with influenza A virus (PR8) and immuno-stained 12 hours later for hemaggutinin (green: hemagluttinin, HA).
  • C Nontargeting siRNA negative control. Magnification, 4x.
  • FIG. 1C is a line graph showing the results of the screen are shown with the siRNA SMARTpools ranked in order of average Z-score, from lowest (decreased infection) to highest (increased infection). The position of known influenza A virus-host factors and several newly identified genes that scored in the screen are indicated.
  • FIGs. 1G, H and I are Western blots for cells in ID, IE and IF.
  • NP siRNA targeting flu nucleoprotein
  • C Non-targeting siRNA negative control.
  • Ran levels are provided to demonstrate relative protein loading when cross-reacting bands were not present.
  • FIG. 3 A is a bar graph showing relative fold induction in U20S cells transfected with the indicated siRNAs for 72 hours, then infected with PR8. Infection was assessed by IF for HA (surface or entire cell), NP or M2, 12 hours after viral addition. Relative fold infection is normalized to non-targeting (C) control.
  • FIG. 3B shows results of U20S cells transfected with the indicated siRNAs and assessed for IFITM3 levels by Western blotting.
  • FIG. 3C is a bar graph showing changes in percent infection when U20S cells stably expressing either IFITM3 with a C-terminal HA-epitope tag (IFTIM3-HA 6R ) lacking the target site for siRNA IFITM3-6, or the vector alone, were transfected with the indicated siRNAs (x-axis). After 72 hours the cells were incubated without (no virus) or with influenza A (PR8) for 12 hours, then stained for HA expression. The anti- hemagglutinin antibody used to detect flu infection does not recognize the HA epitope tag on IFITM3-HA 6R (no virus, uninfected control).
  • FIG. 3D is an image of a Western blot showing U20s cells stably expressing either IFTIM3-HA 6R or the vector alone were transfected with the indicated siRNAs and assessed 72 hours after transfection with the antibodies indicated in the left column.
  • FIG. 3E is an image of a Western blot showing U20S cells that were untreated, or incubated with either IFN- ⁇ , or IFN-a. After 24 hours the levels of IFITM3 were assessed.
  • FIG. 3F is a line graph showing percent infection in U20S cells transfected with the indicated siRNAs, then left untreated or incubated with IFN- ⁇ 48 hours later. After 24 hours of IFN incubation, the cells were infected with increasing amounts of PR8. Twelve hours after infection the cells were stained for HA expression and assessed by IF.
  • FIG. 3G is a bar graph showing that IFITM3 is required for the anti-viral effect of IFN- ⁇ .
  • C Non-targeting siRNA negative control.
  • FIG. 4A is a bar graph showing changes in relative fold infection in A549 cells transduced with retroviruses containing epitope tagged cDNAs for the indicated IFITM proteins, or empty viral vector alone (vector). Two days later the transduced cells were infected with one of the following viruses: influenza A H1N1 PR8 [HI (PR)], influenza A H3N2 A/U
  • 4B is an image showing the results of Western blot analysis of A549 cells transduced with retroviruses containing the indicated IFITM proteins, or the empty vector control virus. After 48 hours the levels of the IFITM protein were checked using anti-C9 antibody, which detects the epitope tag. ⁇ -actin levels show relative protein loading.
  • FIG. 4C is an image showing the results of Western blot analysis of IFITM3 expression in A549 and U20S cells stably over-expressing IFITM3.
  • FIG. 4D is a bar graph showing changes in relative fold infection in A549 cells transduced with retroviruses containing the indicated IFITM proteins, or the empty viral vector (vector). Two days later the cells were incubated with MLV-EGFP virus pseudotyped with the indicated envelope proteins.
  • HI (PR) influenza A virus PR8, H3 (Udorn): H1N1 A/Udorn/72 , H5(Thai): A/Thailand2(SP-33)/2004, H7(FPV):
  • FIG. 4G is a line graph illustrating cell surface expression of N-terminally epitope tagged IFITM3, measured by flow cytometry using the anti-Myc antibody 9E10, is shown for vector and Myc-IFITM3-transduced (IFITM3) A549 cells analyzed in (A and E). Cells were assayed without permeabilization.
  • FIG. 4H shows human IFITM 1, 2 and 3 protein sequence alignment.
  • the alignment was performed with ClustallW. * identical aa, : conservative aa substitution, . semi-conservative substitution.
  • VLPs flaviviral viral like particles
  • EGFP expressing EGFP
  • OMSK Omsk virus
  • FIG. 6C is an image of a Western blot showing the results of an experiment wherein MEFs from 6A) were assessed by Western blot for the presence of Ifitm3 protein. GAPDH levels are provided to show protein loading.
  • FIG. 6D is an image of a Western blot showing the results of an experiment wherein the indicated MEFS were assessed for IFITM3 expression by Western blot. Ran demonstrates protein loading.
  • FIG. 6E is an image of a Western blot showing the results of an experiment wherein MEFs were left untreated (buffer), or incubated with either IFN- ⁇ , IFN-, or PR8 virus. After 24 hours the levels of Ifitm3 were checked by Western blot. Ran levels show relative protein loading.
  • FIG. 6F shows an extended IFITM1, 2, 3 and 5 protein sequence alignment.
  • the alignment was performed with ClustallW. * identical amino acids (aa), : conservative aa substitution, . semi-conservative substitution.
  • FIG. 7 is a schematic model of IFITM-mediated inhibiton of viral infection.
  • IFITM1,2 and 3 are represented by a dual-transmembrane protein. IFITM proteins interfere with influenza A virus infection by preventing viral fusion, thereby stopping vRNPs from gaining access to the host cell's nucleus. Instead invading pathogens are directed to the cell's lysosomes where they are held and destroyed. IFN also inhibited viral fusion and vRNP nuclear translocation, and IFITM3 was required for this activity. These data thus extend our previous work demonstrating the requirement of IFITM3 for 50-80% of IFN' s virustatic effects in vitro, and also reveal that an early IFN-mediated block to infection is occurring at viral fusion [11]. IFITM3 thus represents a previously unappreciated type of anti-viral effector that permits viral entry into the endosomal compartment, but denies egress into the cytosol, thereby neutralizing the cumulative infectious threat to the organism.
  • FIG. 8A is a Western blot of MDCK cells stably expressing either the vector control or IFITM3 transgene.
  • FIG. 8B is an image of a Western blot showing the results of an experiment wherein primary chicken fibroblasts (ChEFs) stably expressing IFITM3 or vector alone were checked for IFITM3 expression.
  • FIGs. 9A-H are lists of a number of exemplary IFITM3 sequences in chickens, chimpanzees, rainbow trout, mice, macaques, horses, dogs, rats, cows, and humans.
  • FIG. 10 is an alignment showing that the influenza A viral strains WS/33, WSN/33, and H3/Udorn possess Critical Anti-IFN Molecular Determinants within their NSl Proteins.
  • the NSl proteins of the three viruses used in this study, PR8 (NCBI Protein database locus link, ACR15353), WSN/33 (ABF83571), WS/33 (AAA21582.1) and H3/Udorn (ABD79037.1) are shown, along with the NSl protein from the highly pathogenic 1918 influenza strain A/Brevig_Mission/l/l 8 for comparison (AAK14368).
  • the amino acids, F 103 and Ml 06 suggested by Kochs et al.
  • FIG. 11 A is a line graph showing the results of experiments wherein A549 cell lines were infected with increasing amounts of VN/04. Twelve hours after infection the cells were immunostained for NP expression and scored for infection status. Values are representative of two independent experiments.
  • FIG. 1 IB shows the results of experiments wherein A549 cell lines were incubated on ice with HlNl WSN/33 to permit viral-host binding. Cells were washed, fixed and immunostained for surface-bound HA protein, then analyzed by flow cytometry. Values given are percentage of cells staining for surface HA. Values are representative of three independent experiments.
  • FIG. 11C is a bar graph showing the results of experiments wherein A549 cell lines were infected with HlNl WSN/33. At the indicated time points cDNA was prepared and viral M2 mRNA expression levels were measured by qPCR. Values were normalized to host cell GAPDH mRNA levels. Values represent the mean +/- SD of three independent experiments.
  • FIG. 12 is a bar graph showing the results of experiments wherein IFITM3 prevented the nuclear translocation of viral genomes to cell nuclei in vitro.
  • MDCK cells stably overexpressing the empty vector control (MDCK-Vector) or IFITM3 (MDCK- IFITM3) were incubated with HlNl PR8 on ice. Warm media was added at time zero. Cells were then fixed at the indicated time points post infection and hybridized with RNA probes against the viral NP genome (NP vRNA PR8) and stained for DNA, then imaged by confocal microscopy.
  • Quantitation of nuclear viral RNA puncta was done using Imaris image analysis software by determining the number of viral RNA puncta per nucleus of the MDCK-Vector and IFITM3 cells at the indicated time points. Values represent the mean +/- the SD of three independent experiments.
  • FIG. 13A is a line graph showing that IFITM3 inhibits fusion of HA
  • FIG. 13B is a bar graph showing that fusion of HA pseudoparticles increases after IFITM3 knockdown.
  • WI-38 primary fibroblasts stably transduced with a short hairpin RNA against IFITM3 (WI-38 shM3), a shRNA control with a scrambled sequence (WI-38 shScr), or the IFITM3 cDNA (WI-38 M3) were exposed to HA pseudoparticles (H1N1) containing BLAM-Vpr.
  • FIG. 14 shows the results of Western blotting of lysates from A549-IFITM3 or A549-Vector cells treated or untreated with IFN-a or ⁇ for 24 h and probed with the indicated antibodies. GAPDH levels are provided to demonstrate relative protein loading. These images are representative of three independent experiments.
  • FIG. 15 shows the sequence of NCBI Reference Sequence: NP_066362.2 interferon-induced transmembrane protein 3 (1-8U) [Homo sapiens], SEQ ID NO:598.
  • FIG. 16 shows the sequence of NCBI Reference Sequence: NP_006426.2 interferon induced transmembrane protein 2 (1-8D) [Homo sapiens], SEQ ID NO:599.
  • FIG. 17 shows the sequence of NCBI Reference Sequence: NP_003632.3 interferon induced transmembrane protein 1 (9-27) [Homo sapiens], SEQ ID NO:600.
  • Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses anti-viral restriction factors to defend against infection.
  • a functional genomic screen was used to identify, in human cells, over 120 influenza A virus-dependency factors (IDFs) with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing.
  • IDFs influenza A virus-dependency factors
  • the screen also led to the discovery that the interferon-inducible trans-membrane proteins, IFITM1, 2 and 3, restrict early replication of influenza A virus.
  • the IFITM proteins control basal resistance to Influenza A, but are also inducible by interferons (IFN) type I and II, and are critical for IFN's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviruses, including dengue virus (DNV) and West Nile virus (WNV). Collectively this work identifies a new family of anti-viral restriction factors, which mediate the cell-intrinsic innate immune system's response to at least three major human pathogens.
  • IFN interferons
  • the screen identified the IFITM proteins as viral restriction factors. IFITM proteins were originally described 25 years ago by Freidman et al. based on their expression in neuroblastoma cells after interferon treatment (Friedman et al, Cell 38, 745- 755, 1984).
  • the IFITMl, 2, 3 and 5 genes lie adjacent to one another on chromosome 11, and all encode for two predicted membrane-spanning domains, separated by a highly conserved intracellular loop (Lewin et al, Eur J Biochem 199, 417-423, 1991, Fig. 6F). IFITMl, 2 and 3 are nearly ubiquitously expressed.
  • IFITM5 displays a more restricted pattern, being expressed primarily in bone tissue (Moffatt et al, J Bone Miner Res. 2008 Sep;23(9): 1497-508). While IFITMl homologs are found in frog, fish, fowl and swine, IFITM2 and 3 homologues are found in more recently diverged mammalian species (mouse, rat, cow, chimpanzee, and human) (see the world wide web at ncbi.nlm.nih.gov/sites/entrez), suggesting that IFITMl is the original ancestral gene, with IFITM2 and 3 arising in a later gene duplication event (Fig. 6F).
  • the IFITM proteins have been ascribed roles in immune cell signaling, cell adhesion, oncogenesis, and germ cell homing and maturation (Smith, R.A., et al, Genes Immun, 2006. 7(2): p. 113-21; Lange, U.C., et al., Mol Cell Biol, 2008. 28(15): p. 4688- 96; Lange, U.C., et al, BMC Dev Biol, 2003. 3: p. 1; Ropolo, A., et al., Biochem Biophys Res Commun, 2004. 319(3): p. 1001-9; Evans, S.S., et al, J Immunol, 1993. 150(3): p.
  • IFITMl has been reported to reside in lipid rafts on the cell surface, where it may play a role in both cell adhesion and immune cell signaling (Bradbury et al, J Immunol 149, 2841-2850, 1992). However, as of now we know of no functional studies clearly demonstrating an additional function for an IFITM protein family member.
  • the IFITM proteins belong to a protein domain super- family, consisting of over 30 proteins, each possessing two trans -membrane domains and an intervening highly conserved intra-cellular loop (pfam04505, CD225, Interferon-induced transmembrane protein).
  • CD225 proteins that have been reported to be expressed in zebrafish, Xenopus, the purple sea-cucumber, and several bacteria.
  • the IFITM protein is at least 95% identical to these reference sequences, e.g., at least 96%, 97%, 98%, 99% or 100% identical.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • IFITM1, 2 and 3 in pathogen restriction has allowed the development of cells and cell lines that are useful for the production of pathogens, e.g., for use in research or in the manufacture of vaccines.
  • the cells and cell lines are those in which IFITM1, 2, and/or 3 (referred to herein collectively as "IFITM"), or their ascribed actions, are specifically deleted, depleted or antagonized.
  • IFITM3 is deleted but at least one other IFITM family member is retained and is functional.
  • viral restriction factors and interferon-inducible proteins/factors are also deleted, e.g., MxA and MxB, in order to further compromise the viral-producer cells from inhibiting viral replication
  • MxA and MxB are also deleted, e.g., MxA and MxB, in order to further compromise the viral-producer cells from inhibiting viral replication
  • IFITM proteins can act as viral restriction factors by inhibiting endocytosis.
  • the three IFITM3 proteins can be used to change the entry and/or decrease infection of any agent that is endocytosed, e.g., any intracellular pathogens such as viruses, bacteria or bacterial toxins that are endocytosed.
  • any agent that is endocytosed e.g., any intracellular pathogens such as viruses, bacteria or bacterial toxins that are endocytosed.
  • the cells, compositions, and methods described herein can be used to produce or inhibit infection with pathogens or toxins which infect host cells A number of different viruses and pathogens are provided herein.
  • influenza A virus including influenza A virus, influenza B virus, and influenza C virus, all strains of influenza viruses infecting humans, birds, pigs, seals and horses, (e.g., influenza A (H1N1 (both A/WS/33 and
  • SLEV Louis encephalitis virus
  • USUV Usutu virus
  • WNV West Nile virus
  • Kunjin virus Yaounde virus
  • YAOV Yaounde virus
  • Kokobera virus group Kokobera virus
  • Kokobera virus Kokobera virus
  • Ntaya virus group Bagaza virus (BAGV)
  • IHV Ilheus virus
  • ITV meningoencephalomyelitis virus
  • Ntaya virus Ntaya virus
  • TMUV Tembusu virus
  • ZIKV Zika virus
  • Yellow fever virus group Banzi virus
  • BBV Bouboui virus
  • EHV Edge Hill virus
  • Jugra virus JUGV
  • SABV Saboya virus
  • SEPV Sepik virus
  • USV Wesselsbron virus
  • Yellow fever virus YFV
  • viruses with no known arthropod vector including: the Entebbe virus group: Entebbe bat virus (ENTV), Yokose virus (YOKV), the Modoc virus group as follows: aba virus (APOIV), Cowbone Ridge virus (CRV), Jutiapa virus (JUTV), Modoc virus (MODV), Sal Vieja virus (SVV), San Perlita virus (SPV), and the Rio Bravo virus group: as named Bukalasa bat virus (BBV), Carey Island virus (CIV), Dakar
  • Bacterial pathogens and their respective toxins that are endocytosed include, but are not limited to: Gram-negative bacteria (e.g., proteobacteria including
  • Enterobacteriaceae e.g., Escherichia coli (e.g., diarrheagenic and hemorrhagic E. coli, including EHEC 0157), Salmonella, and Shigella), Pseudomonads, Diplococcus (e.g., Moraxella), Helicobacter, Campylobacter (e.g., Campylobacter jejuni),
  • Escherichia coli e.g., diarrheagenic and hemorrhagic E. coli, including EHEC 0157
  • Salmonella and Shigella
  • Pseudomonads e.g., Moraxella
  • Helicobacter Campylobacter (e.g., Campylobacter jejuni)
  • Stenotrophomonas e.g., S. maltophilia
  • Bdellovibrio acetic acid bacteria, Legionella
  • alpha-proteobacteria e.g., Wolbachia
  • cyanobacteria spirochaetes , green sulfur and green non-sulfur bacteria
  • Niesseria e.g., N. gonorrhoeae and N. meningitides
  • Rickettsia e.g., Rickettsia prowazekii
  • Moraxella catarrhalis Pasteur ellaceae (e.g., Haemophilus influenzae) ;Chlamydophylla (e.g., Chlamydia psittaci and C.
  • abortus some additional specific examples of gram-negative bacteria include Klebsiella pneumoniae, Bartonella henselae, Legionella pneumophila, Pseudomonas aeruginosa, Ehrlichiosis Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Yersinia pestis and Yersinia enterocolitica, Salmonella typhi, Burkholderia pseudomallei (glanders), Coxiella burnetii (Q fever), Brucella species (brucellosis), Francisella tularensis (tularemia) and Acinetobacter baumannii) and Gram- positive bacteria (e.g., Bacillus, Clostridium, Sporohalobacter, Anaerobacter,
  • Staphylococcus e.g., Group A Staphylococcus aureus
  • Streptococcus Enterococcus
  • Corynebacterium Nocardia
  • Actinobacteria and Listeria
  • Mollicutes e.g., Mycoplasma and Mycobacterium including Mycobacterium Tuberculosis, M. Leprae and Multidrug-resistant Tuberculosis
  • fungi e.g., Coccidioides, e.g., C. posadasii, and Coccidioides immitis
  • protozoa e.g., Cyclospora cayatanensis
  • Cryptosporidia e.g., C.
  • isolated cells that have a specific reduction in an IFITM, plus one or more other viral restriction factors.
  • host cell and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell that was contacted with a nucleic acid molecule (e.g., an inhibitory nucleic acid that reduces expression of an IFITM protein, or a knockout vector that induces functional deletion of one or more IFITM genes from the genomic DNA of the cell), but to the progeny or potential progeny of such a cell that also contain the nucleic acid molecule.
  • a nucleic acid molecule e.g., an inhibitory nucleic acid that reduces expression of an IFITM protein, or a knockout vector that induces functional deletion of one or more IFITM genes from the genomic DNA of the cell
  • the cells are also infected with an intracellular pathogen, e.g., a virus, bacterium, or bacterial toxin, e.g., as described herein.
  • an intracellular pathogen e.g., a virus, bacterium, or bacterial toxin, e.g., as described herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • the cell can be a bacterial cell such as E. coli, insect cells, yeast or mammalian cells (such as African green monkey kidney (Vero), human PER.C6, Madin-Darby canine kidney (MDCK) cells, transformed or primary chicken cells, avian embryonated egg cells, such as chicken, Chinese hamster ovary cells (CHO), HEK 293, or COS cells).
  • Other suitable host cells are known to those skilled in the art. In general, in the methods described herein, the cell will be one that is useful for the production of virus.
  • Vector DNA or inhibitory nucleic acids can be introduced into host cells via conventional transformation or transfection techniques.
  • naked DNA is simply applied to a cell.
  • the vector is a viral vector, known infection protocols can be used.
  • retroviral vectors can be used, e.g., as described in Robertson et al, Nature 323:445-448 (1986). Retroviruses generally integrate into the host genome with no rearrangements of flanking sequences, which is not always the case when DNA is introduced by microinjection or other methods.
  • Cells of the present invention also include those cells obtained from the transgenic animals described herein, e.g., cells from the tissues of those animals that overexpress IFITM 1, 2, or 3.
  • Specific deletion of IFITMl, 2, or 3 can be accomplished using any method known in the art, e.g., using homologous recombination or recombinantly engineered zinc-finger nucleases to delete the selected gene from the genomic DNA, or using inhibitory nucleic acids, e.g., transiently or stably expressed inhibitory nucleic acids.
  • Inhibitory nucleic acids e.g., siRNA, shRNA, miRNA, LNA, antisense, ribozymes, or aptamers, directed against the selected gene(s), can be used to specifically reduce expression of a gene described herein, e.g., IFITM1, 2, or 3, or a gene listed in Table 1 or Table 2.
  • the deletion can thus be permanent (e.g., in the genomic DNA) or transient (e.g., only in the presence of siRNA or antisense).
  • RNA interference is a process that induces the sequence-specific regulation of gene expression in animal and plant cells and in bacteria (Aravin and Tuschl, FEBS Lett. 26:5830-5840 (2005); Herbert et al, Curr. Opin. Biotech. 19:500-505 (2008); Hutvagner and Zamore, Curr. Opin. Genet. Dev.: 12, 225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001); Valencia-Sanchez et al. Genes Dev. 20:515-524 (2006)).
  • RNAi can be triggered by 21 -nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, Mol. Cell. 10:549-561 (2002); Elbashir et al, Nature 41 1 :494-498 (2001)), by microRNA (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase II or III promoters (Zeng et al, Mol. Cell 9: 1327-1333 (2002); Paddison et al, Genes Dev. 16:948-958 (2002); Denti, et al, Mol.
  • siRNA small interfering RNA
  • the methods described herein can use dsRNA molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is complementary to the first strand.
  • the dsRNA molecules can be chemically synthesized, or can transcribed be in vitro or in vivo, e.g., shRNA, from a DNA template.
  • the dsRNA molecules can be designed using any method known in the art. Negative control siRNAs should not have significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.
  • siRNA derivatives e.g., siRNAs modified to alter a property such as the specificity and/or pharmacokinetics of the composition, for example, to increase half-life in the body, e.g., crosslinked siRNAs.
  • the invention includes methods of administering siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked.
  • the oligonucleotide modifications include, but are not limited to, 2'-0-methyl, 2'-fluoro, 2'-0-methyoxyethyl and phosphorothiate, boranophosphate, 4'-thioribose.
  • the siRNA derivative has at its 3 ' terminus a biotin molecule (e.g., a photocleavable biotin), a peptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organic compounds (e.g., a dye such as a fluorescent dye), or dendrimer.
  • a biotin molecule e.g., a photocleavable biotin
  • a peptide e.g., a Tat peptide
  • a nanoparticle e.g., a peptidomimetic
  • organic compounds e.g., a dye such as a fluorescent dye
  • the inhibitory nucleic acid compositions can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life.
  • the conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al, Drug Deliv. Rev.:47(l), 99-1 12 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al, J.
  • inhibitory nucleic acid molecules can also be labeled using any method known in the art; for instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine.
  • a fluorophore e.g., Cy3, fluorescein, or rhodamine.
  • the labeling can be carried out using a kit, e.g., the SILENCERTM siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e.g., using 3 H, 32 P, or other appropriate isotope.
  • a kit e.g., the SILENCERTM siRNA labeling kit (Ambion).
  • the siRNA can be radiolabeled, e.g., using 3 H, 32 P, or other appropriate isotope.
  • Direct delivery of siRNA in saline or other excipients can silence target genes in tissues, such as the eye, lung, and central nervous system (Bitko et al, Nat. Med. 1 1 :50-55 (2005); Shen et al, Gene Ther. 13 :225-234 (2006); Thakker, et al, Proc. Natl. Acad. Sci. U.S.A. (2004)).
  • efficient delivery of siRNA can be accomplished by "high- pressure" delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu (1999), supra; McCaffrey (2002), supra; Lewis, Nature Genetics 32: 107-108 (2002)).
  • Liposomes and nanoparticles can also be used to deliver siRNA into animals. Delivery methods using liposomes, e.g. stable nucleic acid-lipid particles (SNALPs), dioleoyl phosphatidylcholine (DOPC)-based delivery system, as well as lipoplexes, e.g. Lipofectamine 2000, TransYT- ⁇ , have been shown to effectively repress target mRNA (de Fougerolles, Human Gene Ther. 19: 125-132 (2008); Landen et al, Cancer Res.
  • SNALPs stable nucleic acid-lipid particles
  • DOPC dioleoyl phosphatidylcholine
  • lipoplexes e.g. Lipofectamine 2000, TransYT- ⁇
  • Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002), supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. Id. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari et al, Proc. Natl. Acad. Sci. USA 99(22): 14236-40 (2002)).
  • Synthetic siRNAs can be delivered into cells, e.g., by direct delivery, cationic liposome transfection, and electroporation. However, these exogenous siRNA typically only show short term persistence of the silencing effect (4-5 days).
  • Several strategies for expressing siRNA duplexes within cells from recombinant DNA constructs allow longer- term target gene suppression in cells, including mammalian Pol II and III promoter systems (e.g., HI, Ul, or U6/snRNA promoter systems (Denti et al. (2004), supra; Tuschl (2002), supra); capable of expressing functional double-stranded siRNAs (Bagella et al, J. Cell. Physiol.
  • RNA Pol III Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence.
  • the siRNA is complementary to the sequence of the target gene in 5'- 3 ' and 3 '-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs.
  • Hairpin siRNAs, driven by HI or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al. (1998), supra; Lee et al. (2002), supra; Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Sui et al. (2002) supra).
  • Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when cotransfected into the cells with a vector expression T7 RNA polymerase (Jacque (2002),
  • siRNAs can be expressed in a miRNA backbone which can be transcribed by either RNA Pol II or III.
  • MicroRNAs are endogenous noncoding RNAs of approximately 22 nucleotides in animals and plants that can post- transcriptionally regulate gene expression (Bartel, Cell 116:281-297 (2004); Valencia- Sanchez et al., Genes & Dev. 20:515-524 (2006))
  • One common feature of miRNAs is that they are excised from an approximately 70 nucleotide precursor RNA stem loop by Dicer, an RNase III enzyme, or a homolog thereof.
  • a vector construct can be designed to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells.
  • miRNA designed hairpins can silence gene expression (McManus (2002), supra; Zeng (2002), supra).
  • Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to the production of a desired siRNA molecule.
  • Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage, destabilization, and/or translation inhibition destruction.
  • the mRNA to be targeted by the siRNA generated from the engineered RNA precursor will be depleted from the cell or organism, leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism.
  • an “antisense” nucleic acid can include a nucleotide sequence that is
  • the antisense nucleic acid can be complementary to an entire coding strand of a target sequence, or to only a portion thereof (for example, the coding region of a target gene).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the selected target gene (e.g., the 5' and 3' untranslated regions).
  • An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a target mRNA but can also be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the target mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • a "gene walk" comprising a series of oligonucleotides of 15-30 nucleotides spanning the length of a target nucleic acid can be prepared, followed by testing for inhibition of target gene expression.
  • gaps of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested.
  • antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a target protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription, splicing, and/or translation.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.
  • the antisense nucleic acid molecule of the invention is an V-anomeric nucleic acid molecule.
  • An V-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al, Nucleic Acids. Res. 15:6625-6641 (1987)).
  • the antisense nucleic acid molecule can also comprise a 2'-0- methylribonucleotide (Inoue et al. Nucleic Acids Res.
  • the antisense nucleic acid is a morpholino oligonucleotide (see, e.g., Heasman, Dev. Biol. 243 :209-14 (2002); Iversen, Curr. Opin. Mol. Ther. 3 :235- 8 (2001); Summerton, Biochim. Biophys. Acta. 1489: 141-58 (1999).
  • Target gene expression can be inhibited by targeting nucleotide sequences complementary to a regulatory region, e.g., promoters and/or enhancers) to form triple helical structures that prevent transcription of the target gene in target cells.
  • a regulatory region e.g., promoters and/or enhancers
  • Switchback nucleic acid molecule is synthesized in an alternating 5'-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Ribozymes are a type of R A that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art.
  • a ribozyme having specificity for a target-protein encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a target cDNA disclosed herein, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach Nature 334:585-591 (1988)).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a target mRNA. See, e.g., Cech et al. U.S. Patent No.
  • a target mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, Science 261 : 141 1-1418 (1993). Transgenic Knock-in and Knockout Animals
  • non-human transgenic knock-in and knockout animals in which the IFITM 1, 2 or 3 gene(s) is overexpressed or functionally deleted, respectively.
  • Transgenic animals are expected to be resistant to viral and bacterial infection and are therefore useful, e.g., for reducing the incidence and spread of viral infections in the animal population, e.g., in feedstock animals.
  • Knockout Animals are expected to be more susceptible to viral and bacterial infection and thus the animals or cells from those animals can be used for production of the virus or bacterium.
  • knockout chickens can be used to generate IFITM-knockout embryonated eggs for vaccine virus or overall virus production.
  • a “transgenic knock-in animal” is a non-human animal in which one or more of the cells of the animal includes an IFITM 1, 2, and/or 3 knock- in transgene as described herein.
  • a “knockout animal” is a non-human animal in which one or more of the cells of the animal includes an IFITM1, 2, and/or 3 knockout transgene that specifically deletes a functional IFITM1, 2, and/or 3 gene, or disrupts expression of the gene, as described herein.
  • transgenic knock- in and knock-out animals examples include mammals such as rodents (e.g., rats or mice), non-human primates, sheep, dogs, cows, pigs, and goats; birds such as turkeys, chickens, or ducks; amphibians, and the like.
  • the transgenic animals are feedstock animals that are prone to viral infections that can affect humans, e.g., pigs, and poultry, e.g., chickens, turkeys, and ducks.
  • a "transgene” is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and thus remains in the genome of the mature animal, thereby affect the expression of a selected gene product in one or more cell types or tissues of the transgenic animal.
  • Knock-in animals, which include a gene insertion, and knockout animals, which include a deletion of a functional gene or disruption of gene expression are included in the definition of transgenic animals.
  • an "IFITM knock-in transgene” as used herein refers to a construct that includes sequences that have the effect of increasing expression of an IFITM in the cell.
  • the IFITM knock-in transgene includes an IFITM-encoding sequence, and a promoter that drives expression of the IFITM-encoding sequence.
  • the IFITM knock-in transgene includes only an exogenous promoter and optionally additional regulatory sequences to induce overexpression of an IFITM, and flanking sequences that promote homologous recombination into the genome at the site of the IFITM gene, such that the exogenous promoter replaces the endogenous IFITM promoter, and drives expression of the IFITM in the cells.
  • the exogenous promoter is a cell-, tissue-, or timing-specific promoter, e.g., a promoter that will turn on expression of the IFITM transgene in a specific cell or tissue, or at a specific time in development.
  • the exogenous promoter is inducible, and thus can be triggered by the administration of an inducing agent.
  • inducible promoters that can be used in transgenic animals are known in the art.
  • the transgene is generally integrated into or occurs in the genome of the cells of a transgenic animal.
  • IFITM knockout transgene refers to a construct that includes sequences that have the effect of specifically decreasing IFITM1, 2, or 3 expression in the cell.
  • the IFITM knockout transgene disrupts the endogenous IFITM -coding sequence, or disrupts the promoter or other regulatory sequences that drive expression of the IFITM -coding sequence.
  • the IFITM knock-out transgene includes sequences that promote homologous recombination into the genome at the site of the IFITM gene, such that the exogenous promoter replaces the endogenous IFITM promoter, and disrupts expression of IFITM in the cells.
  • the knockout is a cell-, tissue-, or timing-specific knockout, e.g., that disrupts expression of the IFITM transgene in a specific cell or tissue, or at a specific time in development; for example, a cre-lox system can be used that crosses an animal expressing a tissue, cell, or timing-dependent recombinase (e.g., ere) with an animal expressing a floxed IFITM transgene.
  • the knockout is inducible, and thus can be triggered by the administration of an inducing agent.
  • a number of such inducible promoters that can be used in transgenic animals are known in the art, e.g., inducible cre-lox systems.
  • the knockout transgene is generally integrated into or occurs in the genome of the cells of a transgenic animal.
  • the IFITM knock-in or knockout transgene can be used to express or delete the IFITM protein in one or more cell types or tissues of the transgenic animal; expression of the IFITM transgene in a cell results in expression of the IFITM protein.
  • a transgenic animal as described herein is one in which at least one copy of an IFITM transgene has been introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a line of transgenic animals e.g., mice, rats, guinea pigs, hamsters, rabbits, or other mammals
  • Methods known in the art for generating such transgenic animals would be used, e.g., as described below.
  • transgenic animals can be used to generate an animal, e.g., a mouse, chicken, pig, cow, or goat, that bears one IFITM transgene "allele.” Two such heterozygous animals can be crossed to produce offspring that are homozygous for the IFITM transgene allele, i.e., have the sequence encoding the IFITM transgene integrated into both copies of a chromosome.
  • a suitable vector including a sequence encoding or disrupting IFITM1, 2 or 3 is introduced into a cell, e.g., a fertilized oocyte or an embryonic stem cell.
  • a cell e.g., a fertilized oocyte or an embryonic stem cell.
  • Such cells can then be used to create non-human transgenic animals in which said sequences have been introduced into their genome.
  • These animals can then in turn be bred with other transgenic animals that harbor the IFITM3 transgene, or another viral restriction factor, e.g., MxA or MxB.
  • transgenic animals particularly animals such as mice, via embryo manipulation and electroporation or microinjection of pluripotent stem cells or oocytes, are known in the art and are described, for example, in U.S. Patent Nos.
  • a transgenic animal can be made by injecting a vector made as described herein into the pronucleus of a fertilized oocyte and used for generation of a transgenic animal with the IFITM transgene expressed in all cells, using standard transgenic techniques, e.g., as described in "Transgenic Mouse Methods and Protocols (Methods in Molecular Biology),” Hofker and van Deursen, Editors (Humana Press, Totowa, N.J., 2002); U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent Nos. 4,873, 191 and 6,791,006, and in Hogan, "Manipulating the Mouse Embryo," Nagy et al, Editors (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2002).
  • a transgenic founder animal can be identified based upon the presence of the IFITM transgene in its genome, for example by detecting the presence of the IFITM transgene sequences (e.g., IFITM 1, 2, or 3 and/or the exogenous promoter), or by detecting the presence of the IFITM protein, e.g., by detecting overexpression or a tag incorporated into the IFITM transgene.
  • Founder animals can also be identified by detecting the presence or expression of (e.g., the level of expression of) the IFITM mRNA in tissues or cells of the animals.
  • fibroblasts can be used, such as embryonic fibroblasts or fibroblasts derived from the post-natal animal, e.g., the ear of the post-natal animal.
  • a transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a IFITM transgene can further be bred to other transgenic animals carrying other transgenes.
  • IFITM transgenic animals can be bred to animals expressing other viral restriction factors, e.g., MxA.
  • Such animals would have two or more layers of biologic defenses and the virus would have a harder time generating mutations within one virus that could overcome this "combinatorial restriction".
  • both restriction factors are expressed from a single inducible cis acting element, for example a promoter whose transcriptional activity is stimulated by the presence of a small-molecule that permits an activator to bind and induce transcription of the IFITM genes, so that farmers could add the inducing agent to the animals' feed or water to induce the expression of the transgenes, or increase their basal levels, during times of infection or increased risk of infection.
  • a single inducible cis acting element for example a promoter whose transcriptional activity is stimulated by the presence of a small-molecule that permits an activator to bind and induce transcription of the IFITM genes
  • the present invention also provides a method of screening a cell for a variant form of a gene set forth in Table 1 or 2.
  • a variant can be a gene with a functional deletion, mutation or alteration in the gene such that the amount or activity of the gene product is altered.
  • These cells containing a variant form of a gene can be contacted with a pathogen to determine if cells comprising a naturally occurring variant of a gene set forth in Table 1 or 2 differ in their resistance to infection.
  • cells from an animal for example, a chicken, can be screened for a variant form of a gene set forth in Table 1 or 2.
  • a naturally occurring variant is found and chickens possessing a variant form of the gene in their genome are less susceptible to infection, these chickens can be selectively bred to establish flocks that are resistant to infection. By utilizing these methods, flocks of chickens that are resistant to avian flu or other pathogens can be established. Similarly, other animals can be screened for a variant form of a gene set forth in Table 1 or 2. If a naturally occurring variant is found and animals possessing a variant form of the gene in their genome are less susceptible to infection, these animals can be selectively bred to establish populations that are resistant to infection.
  • These animals include, but are not limited to, cats, fish, dogs, livestock (for example, cattle, horses, pigs, sheep, goats, etc.), laboratory animals (for example, mouse, monkey, rabbit, rat, gerbil, guinea pig, etc.) and avian species (for example, flocks of chickens, geese, turkeys, ducks, pheasants, pigeons, doves etc.). Therefore, the present application provides populations of animals that comprise a naturally occurring variant of a gene set forth in Table 1 or 2 that results in decreased or increased susceptibility to viral infection, thus providing populations of animals that are either more or less susceptible to viral infection.
  • livestock for example, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals for example, mouse, monkey, rabbit, rat, gerbil, guinea pig, etc.
  • avian species for example, flocks of chickens, geese, turkeys, ducks, pheasants, pigeon
  • test compounds e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds
  • methods for screening test compounds e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment or prevention of viral infections by increasing IFITM expression, or those test compounds that can be used to antagonize IFITM expression and/or actions.
  • Infections that can be treated or prevented using the compounds identified by these methods include infections with the intracellular pathogens, e.g., viruses and bacteria, described herein.
  • small molecules refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons.
  • small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da).
  • the small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).
  • Any small molecule that inhibits activity of a gene product set forth in Table 1, or similarly increases the activity of a gene product in Table 2, can be utilized in the methods of the present invention to decrease infection.
  • These molecules are available in the scientific literature, in the StarLite database available from the European Bioinformatics Institute, in DrugBank (Wishart et al. Nucleic Acids Res.
  • Preferred small molecules are those small molecules that have IC 50 values of less than about ImM, less than about 100 micromolar, less than about 75 micromolar, less than about 50 micromolar, less than about 25 micromolar, less than about 10 micromolar, less than about 5 micromolar or less than about 1 micromolar.
  • test compounds can be, e.g., natural products or members of a combinatorial chemistry library.
  • a set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.
  • Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1 :60-6 (1997)).
  • a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Patent No. 6,503,713, incorporated herein by reference in its entirety.
  • Libraries screened using the methods of the present invention can comprise a variety of types of test compounds.
  • a given library can comprise a set of structurally related or unrelated test compounds.
  • the test compounds are peptide or peptidomimetic molecules.
  • the test compounds are nucleic acids.
  • test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship.
  • the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds.
  • a general library of small molecules is screened, e.g., using the methods described herein.
  • a test compound is applied to a test sample, e.g., a cell or living tissue or organ, e.g., an eye, and one or more effects of the test compound is evaluated.
  • a test sample e.g., a cell or living tissue or organ, e.g., an eye
  • one or more effects of the test compound is evaluated.
  • the ability of the test compound to increase or decrease the expression or function of IFITM proteins is evaluated.
  • the ability of a test compound to decrease viral infectivity and replication by means of staining for viral protein expression, viral genome production, or progeny virus production (titering assay) can be evaluated.
  • the specificity of this test compounds actions via IFITM proteins could be confirmed in an IFITM null or hypomorphic genetic background.
  • the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein.
  • an animal model e.g., a rodent such as a rat, can be used.
  • a test compound that has been screened by a method described herein and determined to increase expression of IFITM can be considered a candidate compound.
  • a candidate compound that has been screened e.g., in an in vivo model of a disorder, e.g., an animal exposed to the virus, and determined to have a desirable effect on the disorder, e.g., on one or more symptoms of the disorder, can be considered a candidate therapeutic agent.
  • Candidate therapeutic agents once screened in a clinical setting, are therapeutic agents.
  • Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.
  • test compounds identified as "hits” can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein.
  • the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.
  • Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating or preventing viral infections as described herein, e.g., infections with an intracellular pathogen as described herein, e.g., a virus or bacterium as described herein.
  • a variety of techniques useful for determining the structures of "hits” can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy.
  • the invention also includes compounds identified as "hits” by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.
  • Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of a viral infection, as described herein.
  • the animal can be monitored for a change in the disorder, e.g., for an improvement in a parameter of the disorder, e.g., a parameter related to clinical outcome.
  • the parameter is viral load, and an improvement would be a decrease in viral load.
  • the subject is a human, e.g., a human with a viral infection, and the parameter is severity or duration of symptoms associated with the viral infection.
  • the methods described herein include methods for the treatment of disorders associated with infections with the intracellular pathogens, e.g., viruses, bacteria, and bacterial toxins, described herein.
  • the disorder is infection with an orthomyxovirus or flavivirus, e.g., an influenza virus.
  • the methods include administering a therapeutically effective amount of a therapeutic compound comprising and IFITM as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • the methods include administering a therapeutically effective amount of an inhibitory nucleic acid that specifically reduces expression of a gene listed in Table 1 as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • to "treat” means to ameliorate at least one symptom of the disorder associated with the infection.
  • administration of a therapeutically effective amount of a compound described herein for the treatment of an infection associated with an intracellular pathogen as described herein will result in a decreased level, duration, or severity of the infection or one or more clinical symptoms of the disorder.
  • treatment includes treating, inhibiting, or preventing viral or bacterial infection in an animal, including a human.
  • Preventing need not require 100% prevention, but can instead include reducing a subject's risk of developing the infection.
  • An infection can be a viral infection, bacterial infection, fungal infection or a parasitic infection, to name a few.
  • An increase, or decrease or inhibition, of infection can occur in a cell, in vitro, ex vivo or in vivo.
  • the term "infection” encompasses all phases of pathogenic life cycles including, but not limited to, attachment to cellular receptors, entry, internalization, disassembly, replication, genomic integration of pathogenic sequences, transcription of pathogen RNA, translation of pathogen RNA, transcription of host cell mRNA, translation of host cell mRNA, proteolytic cleavage of pathogenic proteins or cellular proteins, assembly of particles, endocytosis, cell lysis, budding, and egress of the pathogen from the cells.
  • a decrease or increase in infection can be a decreaseor increase in attachment to cellular receptors, a decrease or increase in entry, a decrease or increase in internalization, a decrease or increase in disassembly, a decrease or increase in replication, a decrease or increase in genomic integration of pathogenic sequences, an increase or decrease in transcription of viral RNA, a decrease or increase in translation of viral RNA, a decrease or increase in transcription of host cell mRNA, a decrease or increase in translation of host cell mRNA, a decrease or increase in proteolytic cleavage of pathogenic proteins or cellular proteins, a decrease or increase in assembly of particles, a decrease or increase in endocytosis, a decrease or increase in cell lysis, a decrease or increase in budding, or a decrease or increase in egress of the pathogen from the cells.
  • This decrease or increase does not have to be complete as this can range from a slight decrease to complete ablation of the infection, or a slight increase to a very large increase.
  • a decrease in infection can be at least about 10%, 20%, 30%, 40%, 50%, 60, 70%, 80%, 90%, 95%, 100% or any other percentage decrease in between these percentages as compared to the level of infection in a control cell, for example, a cell wherein expression or activity of a gene or a gene product set forth in Table 1 has not been decreased.
  • a decrease in infection can be at least about 10%, 20%, 30%, 40%, 50%, 60, 70%, 80%, 90%, 95%, 100% or any other percentage decrease in between these percentages as compared to the level of infection in a control cell that has not been contacted with a compound that decreases expression or activity of a gene or gene product set forth in Table 1.
  • inhibiting transcription of the gene, or inhibiting translation of its gene product can inhibit expression.
  • activity of a gene product for example, an mRNA, a polypeptide or a protein
  • Inhibition or a decrease in expression does not have to be complete as this can range from a slight decrease in expression to complete ablation of expression.
  • expression can be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or any percentage in between as compared to a control cell wherein the expression of the gene product has not been decreased or inhibited or as compared to the level of infection in a control cell that has not been contacted with a compound that decreases expression or activity of a gene or gene product set forth in Table 1.
  • inhibition or decrease in the activity of a gene product does not have to be complete as this can range from a slight decrease to complete ablation of the activity of the gene product.
  • the activity of a gene product can be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or any percentage in between as compared to a control cell wherein activity of a gene product set forth in Table 1 has not been decreased or inhibited, or as compared to a control cell not contacted with a compound that inhibits the activity of a gene product set forth in Table 1.
  • activity of a gene product can be an activity that is involved in pathogenicity, for example, interacting directly or indirectly, with pathogen, e.g. viral protein or viral nucleic acids, or an activity that the gene product performs in a normal cell, i.e. in a non-infected cell.
  • an activity of the proteins and nucleic acids listed herein can be the ability to bind or interact with other proteins.
  • the present invention also provides a method of decreasing infection by inhibiting or decreasing the interaction between any of the proteins of the present invention and other cellular proteins, such as, for example, transcription factors, receptors, enzymes (for example, kinases, phosphatases, synthases, lyases, hydrolases, proteases, transferases, nucleases, ligases, reductases, polymerases) and hormones, provided that such inhibition correlates with decreasing infection by the pathogen.
  • the present invention also provides a method of decreasing infection by inhibiting or decreasing the interaction between any of the proteins of the present invention and a cellular nucleic acid or a viral nucleic acid. Also provided is a method of decreasing infection by inhibiting or decreasing the interaction, either direct or indirect, between any of the proteins of the present invention and a viral, bacterial, parasitic or fungal protein (i.e. a non-host protein).
  • An increase in infection such as that which occurs with the genes in Table 2 can be at least about a 2, 3, 4, 5, 6, 7 ,8 ,9 ,10, 20, 30, 40 , 50, 60 ,70 ,80, 90, or 100 fold increase, or any amount below, above, or in between these amounts, as compared to the level of infection in a control cell, for example, a cell wherein expression or activity of a gene or a gene product set forth in Table 2 has not been increased.
  • An increase in infection can be at least about a 2, 3, 4, 5, 6, 7 ,8 ,9 ,10, 20, 30, 40 , 50, 60 ,70 ,80, 90, or 100 fold increase, or any amount below, above, or in between these amounts that has not been contacted with a compound that increases expression or activity of a gene or gene product set forth in Table 2.
  • the cells of the present invention can be prokaryotic or eukaryotic, such as a cell from an insect, fish, crustacean, mammal, bird, reptile, yeast or a bacterium, such as E. coli.
  • the cell can be part of an organism, or part of a cell culture, such as a culture of mammalian cells or a bacterial culture. Therefore, the cell can also be part of a population of cells. Also included are stem cells.
  • the cell(s) can also be in a subject.
  • viral infections include but are not limited to, infections caused by RNA viruses (including negative stranded RNA viruses, positive stranded RNA viruses, double stranded RNA viruses and retroviruses) and DNA viruses. All strains, types, subtypes of DNA and RNA viruses are contemplated herein.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a therapeutic compound i.e., an effective dosage depends on the therapeutic compounds selected.
  • the compositions can be administered one from one or more times per day to one or more times per week;
  • treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
  • Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions which include IFITM polypeptides described herein as active ingredients.
  • the compositions will include liposomes or other agents that promote incorporation of the IFITM polypeptide into the cell membranes of the target host cells. Also included are the pharmaceutical compositions themselves.
  • compositions typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical
  • Supplementary active compounds can also be incorporated into the compositions, e.g., antiviral or antibacterial compounds.
  • compositions are typically formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • nucleic acid agents can be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine.
  • methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the mammalian transdermal needle- free vaccination with powder-form vaccine as disclosed in U.S. Patent No. 6, 168,587.
  • intranasal delivery is possible, as described in, inter alia, Hamajima et al., Clin. Immunol. ImmunopathoL, 88(2), 205-10 (1998).
  • Liposomes e.g., as described in U.S. Patent No. 6,472,375
  • microencapsulation can also be used.
  • Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996).
  • the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • test compounds e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds
  • agents that modulate infection with a pathogen e.g., viral or bacterial infection, e.g., infection with influenza A.
  • the methods include selecting one or more target genes or proteins from Table 1 or Table 2. and using known assays to identify test compounds that increase or decrease expression or activity of the selected target gene or protein. For example, a test compound that decreases expression of a gene listed in Table 1.
  • a test compound that increases expression of a gene listed in Table L or decreases expression of a gene listed in Table 2 would be a candidate compound for increasing or promoting viral infection.
  • Compounds that decrease or inhibit viral infection are useful as potential therapeutics for the treatment of viral infections.
  • Compounds that increase or promote viral infection are useful in the production of viruses, e.g., for research or therapeutic purposes (e.g., for gene therapy) or for use in vaccines.
  • small molecules refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons.
  • small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da).
  • the small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).
  • test compounds can be, e.g., natural products or members of a combinatorial chemistry library.
  • a set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity.
  • Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1 :60-6 (1997)).
  • a number of small molecule libraries are commercially available. A number of suitable small molecule test compounds are listed in U.S. Patent No. 6,503,713, incorporated herein by reference in its entirety.
  • Libraries screened using the methods of the present invention can comprise a variety of types of test compounds.
  • a given library can comprise a set of structurally related or unrelated test compounds.
  • the test compounds are peptide or peptidomimetic molecules.
  • the test compounds are nucleic acids.
  • test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship.
  • the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds.
  • a general library of small molecules is screened, e.g., using the methods described herein.
  • a test compound is applied to a test sample, e.g., a cell or living tissue or organ, e.g., an eye, and one or more effects of the test compound is evaluated.
  • a test sample e.g., a cell or living tissue or organ, e.g., an eye
  • one or more effects of the test compound is evaluated.
  • the ability of the test compound to increase or decrease expression or activity of a gene or protein listed in Table 1 or Table 2 can be evaluated; alternatively or in addition, the ability of the test compound to inhibit or decrease viral infection in the cell can be evaluated.
  • the test sample is, or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein.
  • an animal model e.g., a rodent such as a rat
  • Animal models may be particularly useful for validating a compound identified as increasing or decreasing expression or activity of a gene or protein listed in Table 1 or Table 2, e.g., for evaluating the ability of the compound to inhibit (treat) or promote a viral infection in the animal.
  • Animal models useful in evaluating therapeutics for the treatment of viral infections induce mice, ferrets, rats (e.g., cotton rats), pigs, and non-human primates. See, e.g., Barnard, Antiviral Research 82(2):A1 10-A122 (2009).
  • Ability to modulate viral infection can be evaluated, e.g., using immunofluorescence assays that detect changes in viral proteins; viral reporter gene assays where infection results in the activation or expression of a reporter protein, e.g., a fluorescent or other detectable reporter such as green fluorescence protein or beta-galactosidase; or titering assays, e.g., where the supernatant from the cultures involving the experimentally manipulated cells is replica plated in a well-by-well manner onto fresh host cells and the specific infectivity of the viral supernatant determined; or cytopathic effect assays, wherein imaging of nuclei or quantitation of ATP can be used as a readout for the remaining viable cells that have resisted infection by a virus or other pathogen or toxin after exposure or treatment by a test compound (see, e.g., Li et al, Proc Natl Acad Sci U S A.
  • a test compound that has been screened by a method described herein and determined to increase or decrease expression or activity of a gene or gene product listed in Table 1 or Table 2 can be considered a candidate compound.
  • a candidate compound that has been screened, e.g., in an in vivo model of a disorder, e.g., an animal model of viral infection, and determined to have a desirable effect on the disorder, e.g., on viral load, or one or more symptoms of the disorder can be considered a candidate therapeutic agent.
  • Candidate therapeutic agents once screened in a clinical setting, are therapeutic agents.
  • Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and/or derivatized, and formulated with physiologically acceptable excipients to form pharmaceutical compositions.
  • test compounds identified as "hits” e.g., test compounds that increase or decrease expression or activity of a gene or gene product listed in Table 1 or Table 2 in a first screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein.
  • the invention includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.
  • Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating disorders associated with viral infection, as described herein, e.g., influenza.
  • a variety of techniques useful for determining the structures of "hits” can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy.
  • the invention also includes compounds identified as "hits” by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.
  • Test compounds identified as candidate therapeutic compounds can be further screened by administration to a cell or animal model of a disorder associated with an infection, e.g., a viral infection, e.g., an infection with influenza A virus, as described herein.
  • a disorder associated with an infection e.g., a viral infection, e.g., an infection with influenza A virus, as described herein.
  • the animal can be monitored for a change in the disorder, e.g., for an infection.
  • the parameter is decreased viral activity observed in standard cell culture and mouse protection assays as known in the art, in example indirect
  • Evidence of an improvement could include decreases in the levels of viral or bacterial products/antigens or viruses or bacteria themselves in cells or animals challenged with the respective virus or other pathogen, as determined by viral titer on reporter cells or animals, and/or decrease in airway tissue and lung tissue viral/pathogen-induced damage, meninegeal and/or brain tissue inflammation or destruction, in addition an improvement would also be inceased duration of survival, and/or well-being of cells or animals as measured by standard parameters. See, e.g., Mount and Belz, Methods Mol Biol. 2010; 595: 299-318; Barnard, Antiviral Research 82(2):A110-A122 (2009); van der Laan et al., Oxford J. Expert Rev Vaccines.
  • the subject is a human, e.g., a human with influenza, and the parameter is duration or severity of symptoms; an improvement would be a shortening in duration and a lessening of severity of symptoms.
  • Symptoms can include fever, muscle aches, headache, lack of energy, dry cough, pharyngitis (sore throat), and rhinitis (runny nose).
  • Example 1 An siRNA Screen for Influenza A Virus Infection Modifying Host Factors
  • RNA interference permits the exploration of functional host- viral interactions.
  • a recent RNAi screen using insect cells identified 98 D. melanogaster proteins that are required for infection by a recombinant influenza virus (Hao et al, Nature 454, 890-893, 2008).
  • Hao et al, Nature 454, 890-893, 2008 Considering the complexities of host-pathogen relationships and the fact that flies lack many of the basic mechanisms mammals use to fight viral infections, we reasoned that further interactions could be brought to light using a human genome- wide siRNA screen.
  • RNAi-based screen was undertaken on an arrayed library targeting 17,877 genes
  • siRNAs were transiently reverse transfected into human osteosarcoma cells (U20S) cells at a 50 nM final concentration, using a final concentration of 0.32% Oligofectamine (Invitrogen, Carlsbad, CA) in a 384- well format (384 well, black plastic, clear bottomed assay plate, Corning 3712). U20S cells were grown in DMEM (Invitrogen Cat#l 1965) with 10% FBS (Invitrogen).
  • siCONTROL Non-Targeting siRNA #2, Dharmacon D-001210-02), and positive control siRNA against NXF1 (SMARTpool M-013680-01) and NP (Dharmacon custom siRNA siGenome synthesis, see below) were present on each plate.
  • Wells containing either buffer alone, or an siRNA pool directed against Polo like kinase one (PLK1, Dharmacon) were present on all plates transfected. The screen was performed in triplicate. The results are shown in Fig. 1A.
  • siRNA pools were classified as hits (decreased infection) if the average of the triplicate plates showed that the percentage of core positive cells was less than 55% of the plate mean, and cell number was not less than 40% of the mean of the plate. Pools that increased infection by greater than 200% of the plate mean were also selected as hits (increased infection).
  • siRNA pools were considered validated if two or more of the individual oligos scored (55% or less infected cells (decreased infection)) or 150% or greater infected cells (increased infection) as compared to the negative control wells on the plate, in either both part one and two or part two alone, and the cell number was not less than 40% of the average of the negative control wells on the plate.
  • transfections were done with a final concentration of 20 nM siRNA to minimize host cell toxicity while still creating a virustatic hypomorphic state.
  • siRNAs against either NP siRNAs against either NP and the host factor, NXF 1 , an mRNA export protein known to be required for influenza A virus infection (Ge et al, 2003; Hao et al, Nature 454, 890-893, 2008).
  • siRNAs against either NP siRNAs against either NP and the host factor, NXF 1 , an mRNA export protein known to be required for influenza A virus infection (Ge et al, 2003; Hao et al, Nature 454, 890-893, 2008).
  • NXFl Dharmacon SMARTpool M-013680-01
  • Gene Ontology terms (vl587; May 2009) were obtained from the Gene Ontology web page (Ashburner et al, Nat Genet 25, 25-29, 2000) and mapping of terms to genes were obtained from the NCBI Gene database (March 17 2009). This analysis was also applied to KEGG Pathways (Kanehisa et al, Nucleic Acids Res 32, D277-280, 2004), Reactome (Vastrik et al, Genome Biol 8, R39, 2007) and protein interactions. Each pathway, reaction, event or the number of interactions for each proteins were essentially treated as a Gene Ontology term for the purpose of statistical analysis.
  • a map of the viral lifecycle was created by connected keywords. Genes were mapped to these keywords using a database that integrates annotation information from UniProt (Bairoch et al, Nucleic Acids Res 33, D154-159, 2005), KEGG (Kanehisa et al, Nucleic Acids Res 32, D277-280, 2004), Reactome (Vastrik et al., 2007, supra), Gene Ontology (Ashburner et al, 2000, supra), NCBI GeneRIF (Mitchell et al, AMIA Annu Symp Proc, 460-464, 2003) and OMIM Human orthologs were mapped to other species using NCBI HomoloGene (Wheeler et al., Nucleic Acids Res 33, D39-45, 2005) and annotations information from these species was used to infer function of human genes.
  • Protein interactions were obtained from the Human Protein Reference Database (Wheeler et al, 2005, supra), The Biomolecular Interaction Network Database (Bader et al, Nucleic Acids Res 29, 242-245, 2001) and BioGrid (Stark et al, Nucleic Acids Res 34, D535-539, 2006). Protein interactions in human as well as in other species were considered.
  • Influenza A viral infection depends on sialic acid residues on the host cell surface, and depletion of the sialic acid transporter, SLC35A1, decreased infection. Consistent with work showing that influenza virus traffics through both early and late endosomes (Sieczkarski and Whittaker, Traffic 4, 333-343, 2003), the screen confirmed the functional role of two small GTPases, RAB5A (surface internalization to early endosome trafficking) and RAB7L1 (early to late endosome trafficking), for viral infection (Somsel Rodman and Wandinger-Ness, J Cell Sci 113 Pt 2, 183-192, 2000). In agreement with Hao et. al, lowering RAB 10 levels inhibited infection (Hao et al., Nature 454, 890-893, 2008).
  • RABIO regulates the movement of endosomes generated from endocytosis downstream of RAB5 (Chen et al, Mol Biol Cell 17, 1286-1297, 2006; Glodowski et al, Mol Biol Cell 18, 4387-4396, 2007). Consistent with the virus depending on a low pH for fusion, loss of any one of four subunits of the multimeric vacuolar- ATPase proton pump (e.g.,
  • ATP6AP 1, ATP6V0B, ATP6V1G1, ATP6V0E2 impeded infection (Marshansky and Futai, Curr Opin Cell Biol 20, 415-426, 2008).
  • the vRNPs Once released from the endosome, the vRNPs are transported into the nucleus though the NPC (Boulo et al, Virus Res 124, 12- 212007; Buss and Stewart, J Cell Biol 128, 251-261, 1995; Clarkson et al, J Mol Biol 263, 517-524, 1996).
  • Nuclear transport factors recovered in the screen include, NUTF2, NUPL1, NUP88, NUP98, and NUP107.
  • splicing complexes were needed for flu replication, including three components of the U2 small nuclear RNP (snRNP), SF3B1, 2 and 3, and the U2 snRNP- interacting proteins, PRPF8, PTBP 1, and FUS. Flu infection also required several members of the U4/U6.U5 tri-snRNP, including SART1, the human homolog of the yeast splicing factor, snu66p, which recruits the tri-snRNP to the pre-spliceosome (Makarova et al, EMBO J 20, 2553-2563, 2001; Stevens et al, RNA 7, 1543-1553, 2001).
  • Fig. ID Four out of four siRNAs targeting SART1 reduced influenza A viral infection, and decreased SART1 protein levels equivalently (Fig. ID). SART1 depletion resulted in lower levels of HA (surface-expressed and total protein), NP and M2 proteins (Fig. ID). Consistent with its splicing function, SART1 loss affected the levels of the M2 protein to a relatively greater extent than that of HA and NP, based on immunoflourescence (IF) staining. However, the decreased levels of all thee viral proteins, products of both spliced (M2) and unspliced (HA, NP) messages, suggests a general block in viral protein production with loss of SART1.
  • M2 products of both spliced
  • HA, NP unspliced
  • SART1 siRNAs were as follows: S ART 1-1 (Dharmacon D-017283-01 ; Target sequence CCGAAUACCUCACGCCUGA (SEQ ID NO:589)); SART1-2 (Dharmacon D- 017283-02; Target sequence GCAAGAGCAUGAACGCGAA (SEQ ID NO:590));
  • SART1-3 Dharmacon D-017283-03; Target sequence GCUACAAACCCGACGUUAA (SEQ ID NO:591)); and SART1-4 (Dharmacon D-017283-04 (Target sequence
  • COPI vesicular transport complex
  • COP1 coatomer 1
  • p-value le-7
  • COPI directs both retrograde intra-Golgi and Golgi to ER transport (Cai et al, 2007).
  • Depletion of six of seven components of COPI (ARCN1, COPA, COPBl, COPB2, COPG, and COPZ1), inhibited HA surface expression, perhaps by interfering with secretion of the host cell receptor(s) and/or trafficking of HA protein to the cell surface.
  • Three or more independent siR were confirmed in the validation round for COPAl, COPBl, COPG and COPZ.
  • COPBl siRNAs were as follows: COPBl-1 (Dharmacon D-017940- 01; Target sequence CGACACAGCUAUGUUAGAA (SEQ ID NO:9)); COPBl -2 (Dharmacon D-017940-02, Target sequence UAUAAGGUCUGUCAUGCUA (SEQ ID NO: 10)); COPBl-3 (Dharmacon D-017940-03, Target sequence
  • CCUCAUGACUUCGCAAAUA SEQ ID NO: 12
  • COPBl -4 Dharmacon D- 017940-04; Target sequence GCUGUUACCGGCCAUAUAA (SEQ ID NO:l 1)).
  • CALCOC02 siRNAs were as follows: CALCOC02 (Dharmacon D-010637-01, Target sequence GACAAGAUCUUCCCAGCUA (SEQ ID NO:231)); CALCOC02 (Dharmacon D-010637-03, Target sequence GAAGACAACCCGUGAGUAU (SEQ ID NO:230)); CALCOC02 (Dharmacon D-010637-04; Target sequence
  • Protein levels were evaluated in the above using Western analysis as follows. Whole-cell extracts were prepared by cell lysis, equivalent protein content boiled in SDS sample buffer, resolved by SDS/PAGE, transferred to Immobilon-P membrane
  • Rabbit anti-SARTl was from Bethyl (A301-423A); mouse monoclonal anti-COPBl (M3A5) from Dr. Victor Hsu (Brigham and Women's Hospital); Purified Rabbit polyclonal to IFITM3 was from Abgent (Cat #AP1153a, along with the corresponding blocking peptide Cat # BP1153); with an additional independent anti-sera from Abeam (#ab74669); mouse purified polyclonal to CALCOC02 was from Abnova (Cat #H00010241-B01p); mouse monoclonal anti-Ran was from BD Biosciences (610340); monoclonal Anti-HA7 from Sigma-Aldrich (Product code H 3663).
  • Reactome is an expertly- curated resource of human biologic pathways, including the host- viral interactions occurring during influenza A virus infection.
  • RNAi screens To identify potential key intermediates, human and fly host factors detected in the RNAi screens were used to select proteins that are significantly (p ⁇ 0.05) connected given the number of their known interactors. Fourteen proteins were predicted as potentially important in the flu lifecycle, including the RNA helicase DHX15, the nuclear transporter TNP02 and the mRNA surveillance and export protein UPF3A. Such testable hypotheses of possible host-viral "nodes and edges" will likely continue to emerge as comprehensive screening efforts and meta-analyses are completed.
  • D-003747-02 AGACGACUGUUACAAGUUU 207.
  • D-003747-01 GCACCGACCUGGUAAGCAU 208.
  • D-014255-02 CAACGUGUCUGUGUUCCUG 215.
  • D-014255-03 GAGCACGUGUUGUCGCUGC 216.
  • D-014255-04
  • D-031204-04 GCGCAGGCUUCGGCUAUUG 219.
  • D-031204-03 CCAAGAUCGUGCAGGGAUA 220.
  • D-031204-02
  • GAAAUACACUAUACACCAU 226 D-016035-02 GGCAUAAAGGAACGUAUUU 227. D-016035-04 UGGCAUGAAUUGAGUAUUA 228. D-016035-01
  • D-016548-01 GAACAACUUUGCCGAGAGG 235.
  • D-016548-03 AAACAAGGAUCUCCAUUAC 236.
  • D-022933-04 GAAGAAAGAUGGCCCCUCA 239.
  • D-022933-03 UCAAGUCCAUCUCUAAUUC 240.
  • D-027234-03 AGGCCCAGGUCCCAUCUUA 255.
  • D-027234-02 GGGAUGACUCUAUGGGCAA 256.
  • D-027234-04
  • GPKOW 27238 NM_ 015698.3 GAACGGAACUGCCUCAUCA 317. D-015129-01
  • MOCS3 27304 NM 014484.3 GAAGAUCCUCCAGUCCUUA 528. D-006406-04
  • NT5C 30833 NM 014595.1 CAACUGGAGGGAGAUCUUA 536.
  • Example 2 The Genetic Screen Identifies IFITM3 as an Influenza A Virus Restriction Factor
  • IFITM3 interferon-inducible transmembrane protein 3
  • PUSLl PUSLl
  • TPSTl TPSTl
  • WDR33 WDR33
  • IFITM3 Depletion of IFITM3 by each of three distinct siRNAs caused increased infection. Five out of six additional unique siRNAs targeting IFITM3 also increased infection in U20S cells, with the phenotype correlating with the level of IFITM3 depletion (Fig. 3A, 3B).
  • the sequence of the IFITM3 siRNAs were IFITM3-1 (Dharmacon D-014116-13, Target sequence ACGUGUUUCUGGUGCUAAA (SEQ ID NO:569)); IFITM3-2 (Dharmacon D-014116-14, Target sequence
  • AUGGAUAGAUCAGGAGGCA (SEQ ID NO:570)); IFITM3-3 (Dharmacon D- 014116-15, Target sequence UGCUGAUCUUCCAGGCCUA (SEQ ID NO:571));
  • IFITM3-4 (Dharmacon D-014116-16, Target sequence UCGUCAUCCCAGUGCUGAU
  • IFITM3-5 Ambion sl95033, Sense GCCUAUGGAUAGAUCAGGATT (SEQ ID NO:573)
  • IFITM3-6 Ambion S195035, Sense CCCACGUACUCCAACUUCCTT (SEQ ID NO:574)).
  • IFITM3 levels were depleted in human primary lung fibroblasts, a more physiologically relevant host cell (WI-38 cells).
  • cervical adenocarcinoma cells HeLa cells, grown in DMEM (Invitrogen Cat#l 1965) with 10% FBS (Invitrogen)
  • IFITM3 siRNAs newly budded virus from these IFITM3 -depleted cells was also increased > 5 fold in titering assays.
  • Lowering IFITM3 levels similarly increased infection by the influenza A H1N1 viral strain, WS/33, which expresses an NS1 protein that has been suggested to be more virulent than PR8's NS1 (Fig. 10 (Haye et al, 2009; Kochs et al, 2007)).
  • IFITM3-HA 6R Cells stably expressing a C-terminal HA-tagged protein, IFITM3-HA 6R , lacking the 3 '-untranslated region targeted by siRNA IFITM3-6, were created.
  • the coding sequence for IFITM3 was obtained from the Vidal Lab Human Orfeome in pDONR-223, after being fully sequence confirmed as correct with the designated Refseq sequence ( M_021034.2), it was recombined into a Gateway -compatible destination vector with a C-terminal HA epitope tag and a Puromycin selectable gene, using LR-clonase
  • IFITM3 -transducing pseudovirus was generated as previously described (Huang et al, 2006, J. Biol. Chem. 281 (2006), pp. 3198-3203; Huang et al, 2008 J. Virol. 82 (2008), pp. 4834 ⁇ 1843), except that myc-IFITM3 was ligated into the pQCXIX vector (Clontech), and used to generate transducing virus, MVL-myc-IFITM3, and myc-IFITM3-expressing virus was incubated with A549 cells. Cells were washed one hour later, and two days later, challenged with MLV-GFP pseudovirus bearing the indicated entry protein. Entry measured by GFP-expression was measured two days later by flow cytometry.
  • IFITM3-HA 6R Overexpression of IFITM3-HA 6R rescued resistance to the virus in the face of siRNA-mediated depletion of the endogenous protein, further confirming it is an "on target” effect (Fig. 3C, D). Thus, IFITM3 is required for basal levels of cellular resistance to influenza A virus infection.
  • IFITM3 The mRNAs for IFITM3, and the closely related and linked genes, IFITM1 and 2 (50%, and 92% amino acid identity, respectively, Fig. 41, 6F), have been reported to be inducible by both IFN type I (a) and II [ ⁇ (Friedman et al, Cell 38, 745-755, 1984; Lewin et al., Eur J Biochem 199, 417-423, 1991)]. This was confirmed for the IFITM3 protein by Western blot and IF (WI-38 cells, cultured in DMEM (Invitrogen Cat#10569), containing IX MEM non-essential amino acids (Invitrogen Cat#l 1 140, 10 mM stock/lOOX) and 15% FBS, Fig. 3E).
  • Interferon-gamma (Invitrogen Cat #PHC4031) was used at 100 ng/ml, Interferon-alpha A (Invitrogen Cat#PHC4014) was used at 100 ng/ml.
  • Cells were incubated with cytokines for 24 hours prior to viral infection.
  • IFITM3 In unstimulated cells, the majority of IFITM3 resides in the ER (based on co-localization with sialic acid and N-acetylglucosamine-conjugated proteins stained by the plant lectin, wheat germ agglutinin, WGA, S3 A).
  • IFN exposure in contrast, triggers the distribution of IFITM3 in a vesicular pattern throughout the cell.
  • the IFN-induction and localization of IFITM3 was confirmed using additional cell lines (U20S and HeLa), anti-sera, and specificity controls (siR A-targeting and peptide-blocking).
  • IFITM3's functional role in the IFN response was examined. About 2200 cells were plated per well in clear bottom 96 well plates (Corning 3603), one day prior to trans fections. Cells were transfected with siRNAs at 50 nM final concentration and Oligofectamine at 0.4% in DMEM containing 15% FBS. For WI-38 primary fibroblast cells, 6000 cells were plated per well one day prior to transfection in Corning 3603 plates. The following day, transfections were done using Lipofectamine 2000 and 100 nM final concentration siRNA.
  • siRNA-mediated target gene depletion occurred over three days, then cells were challenged with one of the following: influenza A virus (H1N1) A/PR/8/34, influenza A (H1N1) virus A/WS/33, or MLV-GPF pseudoviruses (either HI or VSV-G envelope proteins).
  • Influenza A (H1 1) virus A/PR/8/34 (ATCC VR-1469) and influenza A (H1 1) virus A/WS/33 (ATCC VR- 1520) were propagated and viral infectivity was titrated as previously described (Huang et al, 2008).
  • IFN-alpha or -gamma strongly decreased basal levels of influenza A virus infection in both U20S or HeLa cells (Fig. 3E, 3H, 31).
  • the depletion of IFITM3 profoundly decreased the antiviral actions of either IFN-gamma or -alpha (Fig. 3F, Fig.
  • IFITM3-HA 6R 3H, 31. This effect was most pronounced with increasing amounts of virus, consistent with the saturation of a restriction factor. IFN's protective effects were largely restored with the stable expression of IFITM3-HA 6R , which is resistant to the 3'UTR-targeting siR A, IFITM3-6 (Fig. 3G). Because this is a population of cells stably expressing IFITM3-HA 6R to different levels, it would not be expected to see complete restoration of resistance. These results indicate that IFITM3 is required both for basal levels of resistance, as well as for the heightened defenses elicited by IFN ⁇ and a in vitro.
  • IFITM3 The effects of over-expression of IFITM3, or its paralogs, IFITM1 and 2, on viral infection were evaluated.
  • A549 lung epithelial carcinoma cells grown in DMEM (Invitrogen Cat#l 1965) with 10% FBS (Invitrogen) were transduced with viruses expressing the IFITM 1, 2 or 3 proteins (Fig. 4A).
  • the transduced cells expressing the IFITM proteins demonstrated increased resistance to infection with influenza A viruses PR8 (HI (PR)), or H3N2 A/Udorn/72 (H3 (Udorn), Fig. 4A, B). This restriction was not universal, because IFITM proteins did not inhibit Moloney Leukemia virus (MLV, amphotropic envelope).
  • MLV Moloney Leukemia virus
  • the pseudoparticles universally contain an MLV genome encoding the enhanced green fluorescence protein (EGFP) cDNA, however, each strain is exclusively coated with the envelope proteins from one of the following viruses: influenza A virus (strains HI, H3, H5, H7), vesicular stomatitis virus G-protein (VSV- G), Machupo virus (MACH), or MLV (Fig. 4D). Over-expression of any of the three IFITM proteins blocked infection by all four of the influenza A enveloped
  • pseudoviruses with less restriction seen against VSV-G protein, and no decrement observed on viral entry mediated by the MLV ( ⁇ -retrovirus) or MACH (arena virus) envelope proteins.
  • IFITM3 also potently inhibited additional contemporary virulent strains of influenza A viruses.
  • Fig. 8A depicts a Western blot demonstrating the expression of the exogenous IFITM3 transgene in the MDCK cells. Similarly, profound restriction was seen when IFITM3 was stably over-expressed in primary Chicken fibroblast cells (ChEFs), see Fig. 8B.
  • IFITM3 To complement these gain-of-function results, we depleted IFITM3 with siRNAs in U20S cells, than infected them with pseudoparticles, expressing either influenza A virus receptor (HI (PR)) or VSV-G (Fig. 4E). Consistent with the over-expression data, depleting IFITM3 led to increased infection of the influenza A HI pseudoviruses, with VSV-G entry elevated to a lesser extent, with the more potent of the two siRNAs, IFITM3-6.
  • HI influenza A virus receptor
  • VSV-G Fig. 4E
  • Influenza A virus infection begins with the viral envelope proteins interacting with sialylated glycoproteins on the host cell's surface (Lamb and Krug, 2001). There was no reduction, and even a slight increase, in the levels of either a-2,6-sialic acid (SA) or a-2,3- SA when IFITM proteins were over-expressed, pointing away from a reduction in SA concentration underlying the anti-viral actions of the IFITM proteins (Fig. 4F).
  • SA -2,6-sialic acid
  • Fig. 4F a-2,6-sialic acid
  • IFITM3 Inhibits the Early Replication of West Nile virus and Dengue Virus
  • VLPs viral-like particles
  • pseudotyped viruses each uniquely expressing a unique envelope protein
  • MLV-GFP pseudoviruses have been previously described (Huang et al, 2006; Huang et al, 2008).
  • the level of infection of transduced A549 cells was assessed 2 days later by measuring GFP expression by flow cytometry.
  • the level of infection of siRNA-transfected U20S cells after two days of infection was determined by calculating the percent GPF positive cells by IF using the ⁇ scanning miscoscope, after fixation with 4% PFA and staining of nuclei with Hoechst 33342.
  • the VLPs expressed the envelope proteins of one of three flaviviruses, WNV, yellow fever virus (YFV) or the Siberian hemorrhagic tick-borne Omsk virus (OMSK). These VLPs can undergo a single round of infection, and are produced by transiently expressing the respective envelope proteins together with the WNV structural genes, in cells stably expressing sub-genomic WNV replicons containing EGFP (Yoshii and Holbrook, 2009). As observed with influenza A pseudoparticles, all three VLPs were blocked by over-expression of any of the three IFITM proteins, demonstrating that these restriction factors impede first round infection (Fig. 5A).
  • pseudoparticles expressing the envelope proteins of three arenaviruses were not affected by IFITM protein levels.
  • LCMV lymphocytic choriomeningitis virus
  • LASV Lassa virus
  • MACH MLV retrovirus
  • Infected cells were fixed in 4% PFA and immuno-stained with antibodies detecting viral E-proteins (Chemicon), and imaged by fluorescence microscopy (Zeiss).
  • IFITM3 over-expressing or vector control-A549 or -U20S cells were infected with WNV at an MOI of one.
  • Viral propagation and titration of WNV and DNV were performed as follows: WNV (strain 2741) and DNV serotype 2 (New Guinea C strain) viruses were grown on Vero cells (ATCC# CRL-1586) or C6/30 (ATCC# CRL- 1660) cells, respectively.
  • IFITM proteins restrict the replication of two additional human pathogens, DNV and WNV, and may likely help to limit YFV and OMSK infection, based on the VLP data.
  • Example 6 Deletion of the Murine Ifitm Locus Leads to Increased Influenza A Virus Infection in vitro
  • IFITM proteins display a high degree of inter-species homology (Fig. 6F, IFITM or Ifitm, denote the respective human and mouse genes throughout). Therefore, the role of the IFITM proteins in influenza A infection was evaluated in murine embryonic fibroblasts (MEFs) from a previously reported mouse strain, IfitmDel, which has had all five of its Ifitm genes (Ifitml, 2, 3, 5 and 6) removed by gene targeting (Lange et al, 2008). The IfitmDel mice have been shown to develop normally and to have normal phenotypic characteristics within the parameters previously tested (Lange et al, 2008).
  • the human protein was stably expressed in the IfitmDel -I- cells; the transgene rescued the majority of resistance to influenza A H1N1 virus infection (Fig. 6B, D). Ifiitmi levels increased after either IFN treatment, or viral infection (Fig. 6E). A vesicular staining pattern was observed in the +/+ cells, but not in the -/- cells, using anti-sera raised against Ifiitmi; this pattern was identical to that seen for IFITM3 in the human cell lines tested (Fig. 3H, 31)
  • IFITM3 blocks replication of infectious avian H5N1 influenza A virus
  • IFITM3 protected cells from infection by viral
  • IFITM3 lowered the in vitro replication of VN/04 virus at 12 h post-infection (p.i.) in a stably transduced A549 lung carcinoma cell line, as determined by reduced expression of viral nucleoprotein, NP.
  • A549 cells obtained from ATCC were grown in complete media (DMEM).
  • Example 8 IFITM3 inhibits influenza A viral infection after viral-host binding but before viral transcription
  • IFITM3 Inhibition of viral pseudoparticles by the IFITM proteins demonstrated that the restriction occurred during the envelope-dependent phase of the infection cycle. We therefore undertook experiments to more fully determine where IFITM3 prevents infection.
  • A549 cells transduced with IFITM3 or empty vector were cultured in 6-well plate (1.0 x 10 6 cells/well) and lifted using cell dissociation buffer (Gibco), washed in cold PBS twice.
  • Cells and virus were pre-chilled on ice for 30 minutes and mixed at an MOI of 5.0 and incubated at 4°C for 1 hour in a shaker.
  • the viral supernatant and cells were incubated on ice to permit viral binding but prevent endocytosis, which is a temperature- dependent step. After incubation, Cells were washed five times with ice cold PBS and fixed using 4% formaldehyde.
  • the cells were then probed with anti-HA (monoclonal hybridoma, HA-29) antibody for 1 hour at room temperature, followed by anti-mouse alexaflour-488 conjugated antibody for 1 hour with PBS washes in between.
  • the cells were analyzed by flow cytometry on a BD FACS Caliber machine.
  • A549 cells transduced with vector or IFITM3 was incubated with WSN33 virus in triplicate at 37°C for 1 h (hour) and washed extensively with DMEM media. Cells were collected at Oh, lh, 2h, 3h and 4h post infection, trypsinized, and total RNA was isolated using an RNeasy Kit (Qiagen). cDNA was synthesized from 50 nM RNA with sensiscript (Qiagen) kit using random and oligo dT primers.
  • IFITM3 inhibits influenza A viral infection after viral-host binding but before viral mRNA transcription.
  • Example 9 IFN interferes with vRNP nuclear entry, and IFITM3 is required for this antiviral response
  • Vector a vector expressing an IFITM3 cDNA (IFITM3), or vectors expressing short hairpin RNAs (shRNA) targeting IFITM3 (shIFITM3-3) or a scrambled non-targeting control (shScramble).
  • IFITM3 IFITM3 cDNA
  • shRNA short hairpin RNAs
  • the cells were then washed with complete media twice and fixed with 4% formalin (PFA, Sigma) in D-PBS, then stained for NP and DNA and imaged on a confocal microscope. Image analysis software was used to create an outline of each cell's nucleus (pale lines).
  • vRNPs arrive in the nuclei by 60 to 90 min p.i. in the vector control, shIFITM3-3 (shIFITM3), and in the shScramble cells, with the NP signal increasing through to 240 min.
  • shIFITM3 shIFITM3-3
  • shScramble cells with the NP signal increasing through to 240 min.
  • decreased nuclear and increased cytosolic NP staining was observed in IFITM3 expressing cells, consistent with a block after endocytosis but prior to vRNP nuclear translocation. Since as described above IFITM3 is required for the anti-viral actions of IFN, a companion experiment was performed with IFN-a.
  • IFN-a pretreatment also decreased NP nuclear staining in the WI-38-Vector cells however this block was not as complete nor was it associated with similar levels of cytosolic NP staining as those seen with high levels of IFITM3. Consistent with the gain- of-function data, the depletion of IFITM3 resulted in a decrease in IFN's ability to block vRNP trafficking to the nucleus. Similar results were observed with MDCK and A549 cell lines expressing high levels of IFITM3.
  • NP immunostaining provides a useful read-out for subcellular vRNP distribution
  • MDCK cells stably expressing an empty vector MDCK-vector
  • IFITM3 MDCK-IFITM3, Fig. 12
  • cells were processed and stained for the negative stranded NP vRNA of PR8 using a specific RNA probe set (green). Lysotracker red (LTRed), a lysotropic acidophilic dye (pH ⁇ 5.5), was also added along with the warm media at time zero.
  • LTRed Lysotracker red
  • pH ⁇ 5.5 a lysotropic acidophilic dye
  • vRNAs accumulated in the cytosol of the IFITM3 cells, and co- localized with acidic structures based on their staining with LTRed. Similar levels of retained cytosolic vRNPs were observed in experiments without LTRed (data not shown).
  • the loss of the vRNA signal in the LTRed+ inclusions of the MDCK-IFITM3 cells was consistently observed between 150 and 240 min pi.
  • the vRNAs in the control cells increased in number and moved to the cytosol, consistent with the nuclear export of newly synthesized viral genomes occurring in the course of a normal replication cycle (Lamb, R.A. and R.M. Krug, Orthomyxoviridae: The viruses and their replication. 4th ed. Fields Virology, ed. D. Knipe and P. Howley. 2001, Philadelphia: Lippincott Williams and Wilkins).
  • Example 10 Influenza A virus infection is inhibited prior to membrane fusion by either IFN stimulation or IFITM3 overexpression
  • HrV-1 accessory protein Vpr (BLAM-Vpr) and expressing either HA (H1N1, WSN/33), or VSV-g envelope proteins, were produced by plasmid transfection of HEK 293T cells with an HIV-1 genome plasmid derived from pBR43IeG-nef+ (NIH AIDS Research and
  • Cultures for pseudoparticle fusion assays including stably transduced MDCK cells and WI-38 fibroblasts, were plated in 24-well dishes at 90,000 cells per well. At the time of assay, 0.5 mL of virus stock was added to cells and incubated for 2h at 37°C. IN experiments using bafilomycin Al (Sigma), the inhibitor was added at 37°C for lh prior to incubation with virus. After infection, viral media was then aspirated and replaced with complete media containing the ⁇ -lactamase flourogenic substrate, CCF2-AM (Invitrogen) along with 1.7 ⁇ g/mL probenecid.
  • CCF2-AM Invitrogen
  • BLAM-Vpr Upon viral fusion, BLAM-Vpr enters the cytosol and can cleave CCF2, producing a wave length shift from green to blue in emitted light when analyzed by flow cytometry (see, e.g., Tobiume, M., et al., J Virol, 2003. 77(19): p. 10645-50).
  • MDCK-IFITM3 cells a decrease in both HA- and VSV-g-directed fusion was observed, which was comparable to the block produced by poisoning of the host vacuolar ATPase (vATPase) with the macrolide bafilomycin Al (Baf), thereby preventing the low-pH activation of HA required for membrane fusion.
  • Example 11 IFITM3 is present in endosomes and lysosomes and these compartments are expanded with IFITM3 overexpression or IFN treatment
  • IFITM3 IFITM3 primary antibody rabbit polyclonal (Abgent API 153), the endosomal/lysosomal small GTPase protein Rab7 (Rab7 primary antibody - mouse monoclonal (Abeam 50533)), or the lysosomal protein LAMP1 (Lawe, D.C., et al, J Biol Chem, 2002. 277(10): p. 8611-7).
  • IFITM3 Purified Rabbit polyclonal to IFITM3 was from Abgent (Cat #AP1 153a) Human IFITM1 mouse monoclonal antibody was from Proteintech Group, Inc (Cat# 60074-1); Anti-fragilis (Ifitm3) rabbit polyclonal antibody was from Abeam (Cat # ab 15592), mouse monoclonal anti-GAPDH was from BD Biosciences (Cat# 610340). Rab7 primary antibody - mouse monoclonal (Abeam 50533), The LAMP1 [H4A3] and CD63 [H5C6] antibodies were developed by August, J.T.
  • the cells were incubated at 37°C and 5% C02 for 60 min with either Lysotracker Red DND-99 or acridine orange
  • the MDCK-IFITM3 cells were observed to have the majority of the acidic structures present at their periphery, with a relatively open perinuclear space. Flow cytometric analysis also demonstrated an increase in the LTRed signal in both the MDCK and A549 IFITM3 cell lines when compared to controls.

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Abstract

L'invention concerne l'utilisation d'une protéine transmembranaire 1, 2 ou 3 (IFITM1, 2 ou 3) induite par interféron comme facteur de restriction viral, et des procédés pour l'utiliser afin de produire un virus, des animaux transgéniques exprimant IFITM1, 2 ou 3 exogène, et des procédés de traitement ou d'inhibition d'infections virales en ciblant un gène identifié présentement.
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