Replication of retroviruses in species other than their natural host species can be blocked at multiple steps in the viral lifecycle. In recent years, significant progress has been made in describing mechanisms underlying these restrictions. In this proposal we seek to use and extend these observations to generate an HIV-1 that efficiently replicates in owl monkey PBMC (omPBMC), a first step toward developing a new animal model of HIV-1 infection. No animal model of HIV-1 infection currently exists. Such a model would be useful in the evaluation of HIV-1 therapuetics, in vaccine studies, and in studies of HIV-1 pathogenesis. In preliminary data we show that the entry restrictions of two new world monkeys, the owl monkey (Aotus trivirgatus) and the common marmoset (Callithrixjacchus), but not those of the squirrel monkey (Saimiri sciureus), can be readily overcome. We also observe that marmosets exhibit no post-entry barrier, and that, as has been previously demonstrated, the post-entry barrier in owl monkeys can be overcome through a point mutation in the viral gag gene. We show that the owl monkey cell line, OMK, stably expressing owl monkey CD4 and CXCR4 permits robust viral replication, indicating that no additional restrictions are present in these cells. However, HIV-1 virions assembled in owl monkey PBMC are greatly attenuated in their ability to subsequently infect receptor-expressing OMK cells, and these PBMC express substantially higher levels of APOBEC3G than OMK cells. We show that NL4-3 vif partially down-regulates owl monkey APOBEC3G but is unable to down-regulate marmoset APOBEC3G. These data suggest that APOBEC3 proteins are a critical but surmountable impediment to replication of HIV-1 in owl monkey PBMC. Accordingly, the focus of this proposal is to understand and overcome the partial APOBECS-mediated restriction in owl-monkey cells, and to further adapt HIV-1 for efficient replication in owl-monkey PBMC. Adapted viruses will be further modified in their env and nef genes to enhance viral replication in the face of the owl monkey immune response. Modified virus will be further passaged and used to serially challenge owl monkeys housed at the New England Primate Research Center, where this work will be performed. ? ? ?