Nef-Mediated Resistance of Human Immunodeficiency Virus Type 1 to Antiviral Cytotoxic T Lymphocytes

Major histocompatibility complex class I (MHC-I)-restricted CD8+ cytotoxic T lymphocytes (CTL) have emerged as an important arm of immunity in human immunodeficiency virus type 1 (HIV-1) infection (reviewed in reference 68). Increasing evidence suggests that HIV-1-specific CTL are involved in the control of viremia in acute (7, 33) and chronic (41) infections, long-term nonprogressing infection (24, 47), and perhaps prevention of infection in some highly exposed yet uninfected individuals (34, 50). Other studies have documented the potent antiviral effects of CTL in lysing infected cells (65) and suppressing viral replication in vitro (66). Although these data suggest that CTL play an important role in antiviral immunity, it is not clear why they fail to clear HIV-1 in vivo and what mechanisms HIV-1 may employ to escape from this immunity (5). The HIV-1 accessory gene nef was originally thought to be a negative effector of viral transcription (36, 59). Subsequent studies showed this to be a misnomer and identified multiple roles that the Nef protein may play in infected cells. Downregulation of cell surface CD4 has been the most clearly documented function, and this occurs through direct bridging of the cytoplasmic tail of CD4 to an adapter protein which targets it for endocytosis and degradation in lysosomes (2, 3). Other less clearly described effects of Nef are the enhancement of virion infectivity (8, 38, 57) and modulation of cellular activation (4, 6, 15, 23, 51, 54, 56). Finally, downregulation of major histocompatibility complex class I (MHC-I) molecules has been reported as a potentially important function of Nef that may allow for escape from cellular immunity (10, 55). Nef is presumed to interact indirectly with the cytoplasmic tail of MHC-I A and B (but not C) molecules, leading to endocytosis (9). Although the roles of MHC downregulation and other functions of Nef remain unclear, its importance in the development of AIDS in simian immunodeficiency virus (SIV)-infected monkeys (32) and clinically attenuated disease in a patient cohort infected with nef-defective HIV-1 (40) demonstrate its key importance in the immunopathogenesis of HIV-1 infection. The functional significance of MHC-I downregulation by Nef leading to escape from cellular immunity has been somewhat controversial. To our knowledge, only one published study has evaluated the effects of Nef on the interaction of HIV-1-infected cells with CTL (10). Using HIV-1 reporter viruses and flow cytometric analysis, Collins et al. showed that CD4+ cells acutely infected with nef-deleted virus disappear after exposure to CTL in vitro, in contrast to nef-competent HIV-1-infected cells, which are cleared less efficiently. A caveat to this finding, however, was the late time at which susceptibility to cytolysis of the acutely infected cells was assessed (approximately 2 to 5 days after infection). Given the estimation that the entire replicative cycle of HIV-1 requires only about 2 days in vivo (27, 62), measurement of cytolysis of cells exposed to CTL late after infection (more than 2 days) might not be relevant to the interaction between CTL and infected cells. Depending on the kinetics of Nef-mediated resistance to CTL (presumably through MHC downregulation), CTL could clear infected cells before the effects observed by Collins et al. at the late time points of their assay. Vpr is another accessory protein whose immunopathogenic impact and functions have become increasingly clear. The ability of Vpr to mediate nuclear import of the preintegration complex in growth-arrested cells (26) and induce cell cycle arrest (25, 31, 46, 49) have been well documented. Like Nef, Vpr is not required for viral replication in most culture systems in vitro. Conservation of Vpr in vivo, however, suggests important function(s) of this gene in the pathogenesis of HIV-1 infection (58). A candidate for an important in vivo role would be evasion of cellular immune responses, particularly antiviral CTL. In previous studies, we have developed assays to measure the antiviral activities of CTL, as opposed to standard methods measuring lysis or cytokine release in response to target cells infected with recombinant vaccinia or labeled with synthetic peptides (68). We found that HIV-1-specific CTL clones exert potent antiviral effects on T cells acutely infected with HIV-1 (66, 67). Notably, however, these studies almost exclusively utilized the polyclonal HIV-1 strain IIIB, a long-term passaged laboratory isolate of virus containing multiple clones noted to be defective in accessory gene reading frames, including nef (39). Our earlier work therefore examined the function of CTL in the absence of Nef and other accessory proteins. In this study, we examine the roles of Nef and Vpr in the functional antiviral activity of HIV-1-specific CTL clones. Using the in vitro coculture assay we previously developed (66), we show that the antiviral effect of CTL is markedly diminished by the presence of nef in the infecting virus. Furthermore, this phenomenon is not explained by impaired susceptibility of HIV-1-infected cells to the effector functions of CTL, suggesting that escape is due to diminished recognition by CTL. In contrast to nef, vpr does not interfere with the antiviral function of CTL.