' By the end of this year, it has been estimated that approximately 0.7% of the world's population will be seropositive for human immunodeficiency virus (HIV). Most therapy is directed towards inhibition of viral reverse transcriptase or protease. Although over the last two decades much has been learned regarding the replicative cycle of HIV and the cellular factors involved, there are still gaps in our knowledge that could represent future therapeutic targets. For example, in the mouse entry and post-entry blocks to HIV replication have been circumvented by expressing human CD4, a chemokine co-receptor, and cyclin T1. Rodent cells, however, are still not fully permissive for HIV replication, suggesting additional unknown host factors are required. We mutagenized the partially haploid Chinese hamster ovary (CHO) K1 cell line, performed repeated transductions using HIV-eGFP(VSV G) followed by negative cell sorting, and identified approximately 40 cell clones that are highly resistant to HIV and yet permissive to murine leukemia virus (MLV) infection. All clones of a tested subset were also resistant to simian immunodeficiency virus (SIV) and the block did not appear to be at the level of transcription. In this subset of a dozen clones the phenotype appeared to be recessive and four complementation groups were present, based upon pair-wise cell fusions. In this 2-year application we wish to further characterize these HIV-resistant CHO clones. In the first aim for each clone we will determine whether the phenotype is recessive or dominant. For the recessive clones we will perform pair-wise fusions to determine the overall number of complementation groups. In the second aim we will test representative clones from each complementation group and all dominant clones to determine susceptibility to other retroviruses and a spumavirus, including avian leucosis virus, human foamy virus, bovine immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, and SIV. In the third aim, the same clones will be tested to determine the molecular nature of the block to HIV replication. Early and late reverse transcription products, one and two- LTR circles, and proviral integrants will be quantified, as will the activity of the viral long terminal repeat. At the completion of these investigations, a well-characterized panel of cell lines will be freely available to perform more detailed biochemical or genetic complementation studies. ? ? ?
