The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.

Abstract

SummaryConsiderable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-I nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef + HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nefmutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef + and Nef-viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef-viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact neffor subsequent viral growth. We now provide evidence that the Nef § HIV is more infectious than Nef-HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five-to sixfold more infected cells if Nef + viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef + virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef + and Nef-viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef + infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMCderived Nef + HIV. In summary, these findings demonstrate a consistent positive role for the HIV-1 nef gene in promoting viral infection and replication, and suggest that the basis for this phenotype is the increased infectivity of HIV produced from cells expressing nef. These data suggest that HIV-1 Nef, as previously shown for SIV Nef, may play an important role in establishing a fulminant form of viral infection in vivo.

Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors

“…The difference in infectivity between Nef+ and Nef− virions is more pronounced following infection of freshly isolated T cells which are stimulated after infection than it is following infection of T cells which are activated before infection. 32 Consistent with this, we observed a greater decrease in transduction efficiency with deleted vectors in freshly isolated T cells compared with cells that were pre-stimulated. Hence, the absence of Nef may play a role in the decreased transduction efficiency we observed with deleted vectors in T lymphocytes.…”

Nef Stimulates Human Immunodeficiency Virus Type 1 Replication in Primary T Cells by Enhancing Virion-Associated gp120 Levels: Coreceptor-Dependent Requirement for Nef in Viral Replication

“…R5-tropic clones JR-CSF and nef-defective JR-CSF⌬X were gifts from Jerome Zack (33). The YU2b chimera, containing the BssHII-XhoI fragment of YU2 inserted into the equivalent sites of R7, and YU2b⌬N, containing an end-filled XhoI site within the Nef gene, were gifts from M. Feinberg and were described previously (47).…”

“…The absence of high viral loads associated with Nef-defective virus infection suggests that Nef promotes virus replication in vivo. In vitro, Nef enhances HIV-1 replication in primary T lymphocytes and macrophages (24,47,63,65). Although the precise mechanism by which Nef enhances replication in primary T lymphocytes is unknown, we and others have reported that CD4 downregulation by Nef is strongly linked to HIV-1 replication in primary T lymphocytes, peripheral blood mononuclear cells (PBMC), and tonsillar histocultures (29,43).…”

MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1

“…Nef also has a positive effect on the infectivity of virions (18,19), a function which remains mechanistically unexplained. Although the ability to down-regulate CD4 can contribute to the effect of Nef on infectivity (20), this activity is visible by using CD4-negative producer cells and was shown to be independent from other Nef effects (18,(21)(22)(23), it requires its expression in virus-producing cells and is manifested at an early step of the infection process of target cells (18,22,(24)(25)(26).…”