Background: The HIV-1 virulence factor Nef enhances viral infectivity in single-cycle infection assays and accelerates HIV-1 replication in vitro; however, the mechanism by which Nef exerts these effects has not been determined. It has been reported that the effects of Nef are mediated early after viral entry and before the completion of reverse transcription, as viral DNA synthesis is strongly attenuated during infection by Nef-defective virions. Our previous work has demonstrated that Nef is associated with mature HIV-1 cores, implicating Nef in the regulation of HIV-1 core stability.

Methods: We use electron microscopy and a quantitative, kinetic assay of HIV-1 core disassembly for comparative analysis of HIV-1 core structure and stability in wild-type and Nef-defective particles.

Results: Cores were isolated from wild-type and Nef-defective virions in equivalent yields and exhibited identical stability under a variety of experimental conditions. Biochemical analysis of the ribonucleoprotein complex released upon disassembly of wild-type HIV-1 cores in vitro demonstrated the stable association of Nef with a subviral complex containing NC, IN, Vpr, and viral RNA, despite the loss of ~90% of CA.

Conclusions: Our findings that Nef does not influence core stability, but is associated with the viral ribonucleoprotein complex, are consistent with a specific interaction between Nef and an internal component of the HIV-1 core. They further suggest that virion-associated Nef enhances a step in HIV-1 infection that occurs following dissociation of the viral capsid in the target cell.