Restoration of Wild-Type Infectivity to Human Immunodeficiency Virus Type 1 Strains Lacking nef by Intravirion Reverse Transcription

The nef gene of human immunodeficiency virus type 1 (HIV-1) modulates the viral life cycle in several distinct ways (reviewed in references 10, 17, 22, 38, and 40). The absence of a functional nef gene in virus infecting both humans and rhesus monkeys diminishes viral loads and significantly increases the time of clinical progression of disease (11, 28). Expression of nef as a transgene in mice produces many of the pathological effects seen in AIDS (12, 21). In vitro studies demonstrate that Nef can affect multiple cellular functions that help explain how it modulates pathogenicity. Nef promotes the removal of CD4 molecules from the surface of infected cells (4, 16, 35) and downregulates the surface expression of major histocompatibility complex class I molecules (33, 34). These properties are structurally separate features of the Nef molecule (3, 19). Nef also exerts a profound effect on infected cells by altering cell signaling pathways (9, 23, 49) and inducing chemokine release from infected macrophages (47). One key feature of Nef's influence on pathogenesis is that it can enhance the intrinsic infectivity of viral particles (7, 18, 26, 36, 37, 45). Viruses which have nef deleted are from 4- to 40-fold less infectious than wild-type (WT) HIV-1 in tissue culture systems (7, 37). Enhancement of viral infectivity appears to be linked to an increase in the initiation of proviral DNA (2, 43). However, the exact nature of how Nef enhances infectivity and proviral DNA synthesis remains elusive. Several pieces of evidence from prior studies may be relevant in explaining Nef's ability to enhance proviral DNA synthesis and infectivity. Nef is a virion protein (39) and is present in the viral core (30). Although Nef can be cleaved by viral protease to liberate the C-terminal core domain, this cleavage does not appear to correlate with the ability to stimulate virion infectivity (6). Viruses with nef deleted can be restored to WT infectivity by coexpression of Nef in the virus producer cells but not target cells (2, 7). Pseudotyping of HIV-1 virions by vesicular stomatitis virus G (VSV-G) envelope glycoprotein targets viral entry to the endocytic pathway and suppresses the requirement for Nef as well as sensitivity to cyclosporine (2). Recently, it has been shown that Nef may enhance proviral DNA formation by increasing delivery of virions to the cytoplasm of infected cells (42). This study suggests that Nef may function as an entry factor, which may involve interactions with viral envelope protein. In another recent study Nef+ and Nef− virions were allowed to undergo intravirion fusion by pseudotyping a donor particle with gp160 and a target particle with CD4 (56). Nef+ donor virus could enhance the infectivity of a Nef− target particle after fusion. An interesting observation from this study was that the ability to complement nef was dependent on envelope glycoprotein but did not appear to be acting solely at the level of enhancing membrane fusion. One possible explanation for this observation is that Nef may act by altering the composition of the lipid rafts from which HIV-1 particles bud. Other studies have also implicated Nef in altering the composition of membrane rafts as a possible mechanism for enhancement of infectivity (8, 31). It has been shown that cell surface expression of CD4 during virus production can lead to a reduction in infectivity by blocking Env incorporation (31). Since one of Nef's functions is to downregulate CD4 expression, it can also alter infectivity through such a mechanism. It has also been noted that there is significant overlap in mutations known to affect cell sorting of CD4 and enhancement of infectivity (8, 42). Both functions appear to be highly dependent on a functional dileucine motif of Nef. All of these studies suggest that Nef may function at some level prior to reverse transcription to allow an increase in proviral DNA formation. Disassembly of HIV-1 normally occurs as a part of the entry process and, in contrast to some other enveloped viruses, does not appear to require a pH-dependent step (25). It has been shown that HIV-1 particles pseudotyped with envelope proteins that fuse at low pH do not require Nef for enhanced infectivity (5). One method that can be used to uncouple disassembly from the entry process is treating virions by natural endogenous reverse transcription (NERT) (14, 52–55). In this procedure HIV-1 virions are exposed to buffer containing high concentrations of deoxynucleoside triphosphates (dNTPs) and spermidine. The dNTPs enter the virion via the amphipathic domains of the gp41 on the virion (51). Detailed electron microscopy studies have shown that treatment of HIV-1 virions by NERT results in partial disassembly of the viral core (52). This disassembly also results in disruption of a structure known as the core-envelope linkage (CEL), which is an attachment between the smaller end of the core and the envelope (24). NERT treatment can restore vif deletion viruses to WT infectivity (13), which is important since the Vif protein is also thought to be involved in the formation and stabilization of the early reverse transcription complex. Since NERT treatment can rescue the activity of vif deletion virions and it appears that NERT can induce partial disassembly and initiation of reverse transcription, we were interested in how NERT might affect nef deletion virions. In this study, we have treated Nef+ and Nef− virions to induce NERT. We show that pretreatment by NERT can restore infectivity of nef deletion virions to WT levels.