Nef-Mediated Downregulation of CD4 Enhances Human Immunodeficiency Virus Type 1 Replication in Primary T Lymphocytes

The accessory protein Nef is encoded by the primate immunodeficiency viruses human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. The available evidence indicates that Nef is a virulence factor. Adult rhesus macaques infected with simian immunodeficiency virus with nef deleted are dramatically delayed in their progression to AIDS (29). In humans, a small but significant subset of HIV-infected long-term nonprogressors harbor viruses carrying deletions in nef (30, 40, 49). In vitro, Nef is not required for efficient HIV-1 replication in immortalized T-cell lines (41, 57) but enhances HIV-1 replication in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC), as well as purified macrophages and activated primary T cells (21, 41, 57, 61). Several major distinct activities of Nef have been demonstrated in culture, including downregulation of cell surface CD4 molecules, downregulation of cell surface major histocompatibility complex (MHC) class I molecules, enhancement of viral infectivity, and modulation of T-cell signaling pathways. Of these, CD4 downregulation is perhaps the best understood (48). Nef triggers the rapid endocytosis of cell surface CD4 molecules, resulting in their degradation in lysosomes (2, 50). CD4 downregulation by Nef prevents superinfection (8, 36) and facilitates incorporation of Env protein into virions by preventing Env-CD4 interactions at the time of virus budding. Recent reports have also shown that Nef-mediated CD4 downregulation stimulates HIV-1 production (51) and infectivity (17, 31). However, these conclusions were based on studies of viruses produced in 293T cells or immortalized Jurkat T lymphocytes overexpressing CD4 molecules. The importance of Nef-induced CD4 downregulation for HIV-1 replication in primary T cells has not been determined. Nef also enhances HIV-1 infectivity. Wild-type HIV-1 particles produced in CD4-negative COS, HeLa, or 293T cells are 4- to 40-fold more infectious than those with nef deleted in single-cycle infection assays (1, 4, 13, 42, 45). This activity of Nef is independent of CD4 expression in either the producer or target cells, as HIV-1 particles pseudotyped with amphotropic murine leukemia virus envelope, which does not use cellular CD4 as a receptor, also require Nef protein for efficient infectivity (4, 42). Recent studies suggest that Nef may function to enhance infectivity at the level of virus-cell fusion (54), potentially by modifying the viral lipid envelope during HIV-1 assembly (68, 69). To evaluate the relative contributions of CD4 downregulation and CD4-independent infectivity enhancement to Nef-enhanced HIV-1 replication, we first established a purified CD4+-T-cell culture system in which a strong and reproducible effect of Nef on HIV-1 replication is exhibited. CD4+ primary cells were prepared through positive selection by using anti-CD4 antibody-conjugated Dynabeads as previously described (62). This purification protocol typically resulted in >99% purity of CD4+ T lymphocytes, as determined by fluorescence-activated cell sorter (FACS) analysis following staining with CD3- and CD4-specific monoclonal antibodies (data not shown). Immediately following purification, purified T cells were stimulated with staphylococcal enterotoxin B (100 ng/ml; Sigma), a bacterial superantigen, and mitomycin C-killed dendritic cells (1:10 dendritic-cell/T-cell ratio) and cultured for 6 to 8 days prior to infection in medium containing interleukin-2 (250 U/ml). This method of activation, compared to PHA stimulation, more closely mimics antigen-specific stimulation of T cells. For replication assays, cultures of CD4+ primary cells (5 × 104) in 200 μl of medium were inoculated with HIV-1 stocks (1 ng of p24) in medium containing interleukin-2. Every 2 days following inoculation, one-half of the culture medium was removed for quantification of virus production by p24 enzyme-linked immunosorbent assay (64) and an equivalent volume of fresh medium was added. In this culture system, we observed strong and highly reproducible attenuation of the nef-null mutant virus relative to wild-type HIV-1, which replicated efficiently, yielding p24 concentrations of up to 1,000 ng/ml (Fig. ​(Fig.1A1A). FIG. 1. Replication of nef point mutant viruses in primary human T cells. Activated CD4+ T cells were inoculated with wild-type HIV-1 and nef mutant HIV-1 (1 ng of p24 antigen), and supernatants from cell cultures were sampled every 2 days following inoculation ...