Mammalian Genes Involved in Viral Infection and Tumor Suppression

Scientists at Vanderbilt developed a method of identifying of genes, which are necessary for viral growth in cells but nonessential for cellular survival, as well as the methods of treating viral infections based on modification of the function of such genes. The identification of such genes involves the creation of random mutations in single cellular genes by a method which allows the subsequent identification of the mutated gene; selection of cells which remain virus-free after exposure to virus by a method for selectively eliminating persistently infected cells; and subsequent identification of the single mutated gene which precluded viral infection. This invention can be used for identification of host proteins critical for single or multiple virus infection, identification of new molecular targets for antiviral agents and screening of novel antiviral agents.

Clinical Background
• Infectious diseases affect the health of people and animals around the world, causing serious illness and death. Some of the most feared, widespread viruses or bacterial toxins cause devastating human diseases.  The way in which they produce disease is by interfering with normal cellular activity.  The types of agents include viruses, such as influenza, poliomyelitis, smallpox, Ebola, yellow fever, measles and AIDS, and toxin produced by the diarrhea causing bacteria, Clostridium difficile. Also, as highlighted by newly emerging human viruses, such as those producing MERS and SARS, threats abound for agents not covered by existing vaccines or treatments.
• It is noted that vaccines have been effective to prevent important childhood diseases, however relatively few vaccines are available or are not wholly effective, against many human pathogens.  Furthermore, vaccination takes time to be effective and is highly specific for the insulting pathogen.  HIV infection has shown the limits of vaccination to rapidly mutating viruses and cannot anticipate emerging viruses or new bioterrorist-designed viruses.
• Traditional treatments for viral infection include drugs aimed at specific virus components of the pathogen, such a virus manufactured proteins.  Many such drugs have lost potency in combating HIV as the virus proteins have changed in response to the drugs, and some host derived barriers to infection, such as interferon, have never been particularly effective. Thus, the vast majority of viruses lack an effective drug. Those drugs that exist have several limitations and drawbacks that including limited effectiveness, toxicity, and high rates of viral mutations which render antiviral pharmaceuticals ineffective.
• Therefore, an urgent need exists for alternative treatments for viruses and other infectious diseases, and methods of identifying new drugs to combat these threats.
• Bacterial toxins, such as the Clostridia difficile B-toxin have had a spotty history in terms of control and current therapies sometimes resort to transferring feces from non-infected to infected persons to try to halt the toxin damage when other therapy fails.

Technology Description & Novel Features
• Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. Many cellular genes are known to participate in all phases of viral life cycles including attachment to cellular receptors, internalization, disassembly, translation of mRNA, assembly and egress from the cells (Figure 1).  Bacterial toxins may use the same pathways to induce cell death.

Figure 1. A model of the life cycle of reovirus: proposed checkpoints based on function of the cellular genes identified by the insertional mutagenesis.   Several different pathways are shown that are involved in the replication strategy of many viruses types. The genes identified are those that have been trapped are represent potential targets for intervention.ech.

 Description & Novel Features (continued)
• A technology called gene trap insertional mutagenesis has been developed to explore those genes, which are necessary for viral growth in cells but nonessential for cellular survival. The process involves infection of a mammalian cell with a retroviral vector that integrates into the host cell chromosome with no site specificity, so the gene trap vector is expected to disrupt expression of the cellular gene inserted. by this method. A gene trap library of cells with all different mutations could be generated by this method.     
• The mutated library of cells will be challenged with virus infections, genes mutated by gene entrapment may confer virus resistance as a result of either haploinsufficiency or loss of heterozygosity. Resistant clones will be selected and identified by characterizing the genes disrupted by the entrapment vector.
• Over 100 genes has been identified as required for virus infection and may provide cellular targets for new antiviral therapies. Libraries of mutant cells are also available that can be used to screen for resistance to viral infection.

Patent Status
• Cell Lines and Host Nucleic Acid Sequences Related to Infectious Disease, U.S.7,927,793 (Tech ID# VU0426A) 

• ADAM10 and Its Uses Related To Infection, U.S. 8,247,451 (Tech ID# VU0682) 

• Genes Involved in Pox Infection, patent pending (Tech ID# VU0737) 

• Mammalian Genes Involved in Infection, patents pending (Tech ID# VU0974)

• Mammalian Genes Involved in Clostridium Difficile Toxin B Toxicity, patent pending (Tech ID# VU10108)

• Mammalian Genes Involved in Viral Infection, U.S. 7,964,346 (Tech ID# VU9817)

• Mammalian Genes Involved in Viral Infection and Tumor Suppression, U.S. 6,777,177 (Tech ID# VU981)

• Mammalian Genes Involved in Viral Infection and Tumor Suppression, U.S. 7,691,599 (Tech ID# VU9817) 

List of selected publications
Sheng J, Organ EL, Hao C, Wells, KS, Ruley HE, Rubin DH. 2004. Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection. BMC Cell Biology, 5:32 (27 August 2004)

Organ E, Sheng J, Ruley E, Rubin DH. Discovery of Candidate Mammalian Genes That Participate in Lytic Virus Infection, BMC Cell Biology 2004, 5:41 (2 Nov 2004)

Murray JL, Morey NJ, Yilla M, Sheng J, Bellini WJ,  Le Doux JM, Shaw MW, Luo C-C, Sanchez A, Rubin DH, Hodge TW The Rab9 GTPase is Critical for the Replication of HIV-1, Filoviruses, and Measles Virus. J Virol. 2005, Sep; 79(18): 11742-51

Rubin DH, Ruley HE.  Cellular genetics of host susceptibility and resistance to virus infection. Crit Rev Eukaryot Gene Expr. 2006;16(2):155-70.

Ivie SE, Fennessey CM, Sheng J, Rubin DH, McClain MS, 2011 Gene-Trap Mutagenesis Identifies Mammalian Genes Contributing to Intoxication by Clostridium perfringens ε-Toxin. PLoS ONE  6(3): e17787.

Friedrich, BM, Murray JL, Li G, Sheng J, Hodge TW, Rubin DH, O’Brien WA, Ferguson MR, A Functional Role for ADAM10 in Human Immunodeficiency Virus Type-1 Replication in Human Macrophages. Retrovirology, 2011, 8:32 7420

Murray JL, McDonald NJ, Sheng J, Shaw MW, Hodge TW, Rubin DH, O’Brien WA, Smee DF. Inhibition of Influenza A Virus Replication by Antagonism of a PI3K-AKT-mTOR Pathway Member Identified by Gene Trap Insertional Mutagenesis. Antiviral Chemistry & Chemotherapy Antivir Chem Chemother. 2012 May 14;22(5):205-15

Dziuba N, Ferguson NR, O’Brien WA, Sanchez A, Prussia AJ, McDonald NJ, Friedrich BM, Li G, Shaw MW, Sheng J, Hodge TW, Rubin DH, and Murray JL. Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1. AIDS Research and Human Retroviruses. October 2012, 28(10): 1329-1339.

Fennessey CM, Sheng J, Rubin DH, McClain MS (2012) Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2. PLoS ONE 7(10): e46866.

Donald RubinThomas HodgeD. Borden LacyEdward OrganRaymond DuBoisRobert Jeffrey HoganMichelle LaFranceNatalie McDonaldJames Murray
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Janis Elsner

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