Engineering simian-compatible HIV-1

PROJECT SUMMARY/ABSTRACT Construction of infectious and pathogenic HIV-1, or HSIV -- an essentially HIV-1 backbone having minimal SIV gene fragments -- for the simian model will bring tremendous value to studies of AIDS pathogenesis and to vaccine and therapeutic reagents. Our preliminary data and previous findings on SHIV demonstrate that not the Env and receptor/co-receptor molecules, but the inability of the entered HIV-1 to activate the initially resting lymphocytes is the major culprit for the restriction of HIV-1 replication in monkey cells. Nef of SIVpbj1.9 (SNef), but not HIV-1, can sufficiently activate T cells such that the entered SIVpbj1.9 replicates robustly in the resting T cells, causing the infected monkey to die of AIDS within a few weeks. We accordingly constructed chimeric nef, HSNef, which harbors the 5’ 309 nucleotides (nts) of SIVpbj1.9 nef in place of the 5’ 221 nts of HIV-1 nef, non-overlapping region with the long terminal repeat (LTR), and first tested whether the recombinant Nef can activate T cells. Our data showed that HSNef itself sufficiently activated resting T cells, manifested by IL2 generation and Nef-triggered IL2-promoter activity, while HIV-1 Nef (HNef) did not. Further, remarkably, infection of HSIVnef -- which harbors all functional HIV-1 genes, except a small fragment of the SIVpbj1.9 nef, HSNef, within an HIV-1 provirus -- into resting human peripheral blood mononuclear cells (hPBMC) integrated its provirus into host chromosomes without mitogenic activation and thereby replicated in the initially resting hPBMC, whereas HIV-1 could not. These data demonstrated that the small fragment of SIVpbj1.9 nef in HIV-1 backbone was sufficient for replication of the entered HIV-1. Moreover, consistent with the SHIV replication studies in monkeys, HSIVnef comprising HIV-1 Env replicated robustly in activated monkey PBMC (mPBMC), again confirming that Env is not a restriction factor for HIV-1 entry into simian host. These data fundamentally illuminate the molecular function of Nef in pathogenicity for development of the HIV-1/simian AIDS model.