Walker, William H.

ABSTRACT HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non- vif -permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that vif -intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δ vif and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log 10 in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.

Chromosomal integration enables human immunodeficiency virus (HIV) to establish a permanent reservoir that can be therapeutically suppressed but not eradicated. Participation of cellular proteins in this obligate replication step is poorly understood. We used intensified RNA interference and dominant-negative protein approaches to show that the cellular transcriptional coactivator lens epithelium–derived growth factor (LEDGF)/p75 (p75) is an essential HIV integration cofactor. The mechanism requires both linkages of a molecular tether that p75 forms between integrase and chromatin. Fractionally minute levels of endogenous p75 are sufficient to enable integration, showing that cellular factors that engage HIV after entry may elude identification in less intensive knockdowns. Perturbing the p75-integrase interaction may have therapeutic potential.