Background: The Nef protein plays a crucial role in primate lentiviral replication and pathogenesis. By studying an CXCR4-tropic viral clone containing point mutations in the nef gene, we previously demonstrated a strong correlation between CD4 down-regulation by Nef and the ability of Nef to enhance HIV-1 replication in highly purified primary T-lymphocytes. In additional studies, we observed that an CCR5-tropic viral clone replicates efficiently even in the absence of Nef expression. Therefore, we hypothesize that this requirement for Nef for HIV‑1 replication depends on the co-receptor usage of the viral envelope protein.

Methods: We performed replication assays using a purified human T-lymphocyte model system for comparative analysis of the replication fitness of cloned viral isolates and chimeric viruses differing only in the V3 loop region of Env. Using CD4⁺ Hela reporter cells (P4), we also analyzed the infectivity of these viruses produced in 293T and of progeny virions released from primary T-cells.

Results: Nef-defective HIV-1 replicated efficiently when the env gene was specific for CCR5. Single-cycle infection assay of virus released from primary T-cells revealed that the infectivity of X4-tropic HIV-1 was more dependent on Nef than that of R5-tropic virus. In contrast, X4 and R5 viruses released from CD4-negative 293T cells exhibited an identical requirement for Nef. In additional experiments, we observed that the infectivity of infection by R5-tropic HIV-1 was less sensitive to inhibition by soluble CD4 relative X4-tropic viruses.

Conclusions: Our data demonstrate that the requirement for Nef in HIV-1 replication in primary T-cells is dependent on the co-receptor used for viral entry. Our studies further suggest that the specific requirement of Nef for replication of X4-tropic HIV-1 is due to an enhanced sensitivity to CD4-mediated envelope interference.