Identification of Cellular Genes with anti-HIV Activity

S. Valente1, G. Towers2, G. Gao3 and S.P.  Goff1

1Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York (USA); 2Wohl Virion Center, Department of Immunology and Molecular Pathology, University College of London, London (UK); 3Institute of Microbiology, Chinese Academy of Sciences, Beijing (China)

Vertebrate evolution has taken place against a background of constant retrovirus infection.  Several host genes have arisen to control retrovirus replication; these gene products or restriction factors include the Fv1 (Friend-virus-susceptibility-1) gene in mice, Lv1 (Lentivirus susceptibility-1) in non-human primates and Ref1 (restriction factor-1) in humans, that apparently act to inhibit reverse transcription or integration of viral DNA. A great number of antiviral proteins are also induced by interferon, e.g., double stranded RNA activated protein kinase (PKR), 2'5' oligoadenylate synthetases (OAS) or Mx proteins. There is still little understanding of how restriction factors actually protect the cell from retroviral infection; e.g., it’s still not well understood why HIV-1 replicates so inefficiently in monkeys. The identification of new restriction factors and elucidation of their mechanism of action is important and may be useful for therapeutic purposes. This work focuses on the identification of new human cellular genes that restrict the HIV early replication phase. We are currently screening mammalian cDNA libraries for genes conferring resistance to pseudotyped genetically marked retroviruses. Several libraries of randomly primed cDNAs: derived from U937 cells, Owl Monkey cells, as well as from HeLa cells stimulated by interferon are being analyzed. The search for resistance genes is performed the very susceptible lymphocyte cell line, CEM. The strategy developed to select and recover an antiviral gene in the library includes: 1) transduction of CEM cells with a pool of viruses carrying the cDNA library; 2) selection of cells over expressing the cDNA’s of the library; 3) challenging these cells by repeated infection with an HIV retroviral particle expressing the herpes simplex TK suicide gene pseudotyped with a pan-tropic vesicular stomatitis virus envelope protein (VSV-G); 4) elimination of TK positive cells by growth of cells in the presence of the toxic nucleoside analogue Ganciclovir and 5) recovery of the few TK-negative clones as candidate virus resistant cells; the survivors are retested using VSV-HIV particles expressing GFP or puromicinR gene. Results of the current screens will be presented.