Vif Protein

The HIV-1 Vif protein is a 23-kD protein encoded downstream from the pol gene (Rabson et al. 1985; Kan et al. 1986; Lee et al. 1986; Sodroski et al. 1986). All known lentiviruses except EIAV make a similar protein (Oberste and Gonda 1992; Myers et al. 1994; Wieland et al. 1994). Vif is found in association with membranes of infected cells (Guy et al. 1991; Michaels et al. 1993; Goncalves et al. 1994) through interactions with carboxy-terminal basic regions (Goncalves et al. 1995). Mutations in vif result in low viral titers (Fisher et al. 1987; Strebel et al. 1987). Similar results have been reported for the analogous protein in visna virus, the Q gene product (Audoly et al. 1992). The vif mutant phenotype is apparent in some cell types (including peripheral blood mononuclear cells) but is not apparent in others (such as some transformed CD4 T-cell lines), suggesting that some cells can provide a compensatory cellular factor (Fan and Peden 1992; Gabuzda et al. 1992; Michaels et al. 1993; von Schwedler et al. 1993). Expression of the vif mutant phenotype is dependent on the cell where the virus is produced and not on the cell that is the target of infection (Fan and Peden 1992; Gabuzda et al. 1992; Blanc et al. 1993; Sakai et al. 1993; von Schwedler et al. 1993), implying that it is necessary for the production of infectious virus.

Vif has been reported to lead to increased incorporation of the Env protein into virions (Sakai et al. 1993; Borman et al. 1995). Expression of vif during virion formation also leads to more efficient viral DNA synthesis during the subsequent round of infection (von Schwedler et al. 1993; Sova and Volsky 1993). Nonhomogeneous packing of the viral core has been noted for viral particles made in the absence of Vif (Höglund et al. 1994). Higher levels of unprocessed and partially processed Gag precursors are seen with vif mutants (Borman et al. 1995; Simm et al. 1995); however, other workers have observed the vif mutant phenotype without defects in processing, suggesting that these two phenomena are separable (Fouchier et al. 1996; Bouyac et al. 1997). Although earlier studies were unable to find significant amounts of Vif in virions, Vif has more recently been detected in viral particles in concentrations comparable to those of the pol gene products (Liu et al. 1995; Camaur and Trono 1996; Fouchier et al. 1996; Karczewski and Strebel 1996). However, since Vif can also be incorporated into MLV particles, the basis of specificity is not clear (Camaur and Trono 1996).

How Vif exerts its effect on Gag processing is unknown. Perhaps an inappropriately formed core is unable to complete DNA synthesis efficiently during the subsequent round of replication after production of mutant vif ⁻ virus. An alternative function for Vif has been suggested by the observation that it is associated with the cytoskeleton, perhaps mediating interactions between the cytoskeleton and other virion components either during assembly or after infection (Karczewski and Strebel 1996).

Vpr and Vpx Proteins

vpr and vpx are two related genes that encode small proteins (on the order of 120 amino acids long). Both genes are present in HIV-2, closely related SIV strains, and SIVagm, whereas only the vpr gene is present in HIV-1 and most other SIV strains (Guyader et al. 1987; Wong-Staal et al. 1987; Tristem et al. 1992; Myers et al. 1994).

Vpr is a virion-associated protein present at concentrations similar to those of the Gag proteins (Cohen et al. 1990a; Yu et al. 1990; Yuan et al. 1990). Vpr is packaged into viral particles through an interaction with Gag, and deletions of the carboxy-terminal p6 domain prevent incorporation of Vpr (Lu et al. 1993; Paxton et al. 1993; Lavallee et al. 1994). In addition, transfer of p6 to MLV or ASLV Gag confers the ability to encorporate Vpr (Kondo et al. 1995; Lu et al. 1995). A specific motif has been identified within p6 that mediates virion incorporation of Vpr (Lu et al. 1995; Kondo and Göttlinger 1996). Both Vpr and Vpx are directed to the inner face of the cytoplasmic membrane in the presence of the homologous Gag protein (Kappes et al. 1993; Lu et al. 1993).

The effect of vpr or vpx mutations on viral replication in vitro has been variable (for discussion, see Paxton et al. 1993). The presence of these proteins in the viral particle suggests that they may function during an early stage of viral replication. In this regard, Vprhas been shown to localize to the nucleus (Lu et al. 1993; Zhao et al. 1994), and virion-associated Vpr has been implicated in nuclear localization of the viral replication complex in nondividing cells (Heinzinger et al. 1994). Vpr has also been shown to have some activity as a transcriptional activator and an activator of viral replication (Cohen et al. 1990b; Levy et al. 1995) and to induce differentiation in a rhabdomyosarcoma cell line (Levy et al. 1993). Expression of vpr either alone or in the context of HIV-1 replication is sufficient to arrest cells in G₂/M (Rogel et al. 1995; Jowett et al. 1995) by preventing activation of the p34^cdc2–cyclin B kinase complex (He et al. 1995; Jowett et al. 1995; Re et al. 1995); the role of this arrest in viral replication is unclear, although it has been suggested that such an arrest might optimize viral production, block apoptosis, or reduce clonal expansion of infected T cells. An amino-terminal domain of Vpr is important for virion targeting and nuclear localization (Di Marzio et al. 1995; Mahalingam et al. 1995a,b; Yao et al. 1995), whereas a carboxy-terminal domain is important for mediating G₂ arrest (Di Marzio et al. 1995). Vpr can bind the host uracil DNA glycosylase (UNG), but this interaction is not involved in G₂ arrest (Selig et al. 1997).

Vpx is also a virion protein (Franchini et al. 1988; Henderson et al. 1988a; Kappes et al. 1988; Yu et al. 1988). A role for the carboxyl terminus of Gag in the incorporation of Vpx has been demonstrated (Wu et al. 1994), although an interaction between Vpx and p27 CA has been reported in the virion (Horton et al. 1994). However, Vpx does not appear to be a complete functional homolog of Vpr in that Vpx does not induce a G₂ arrest (Di Marzio et al. 1995; Re et al. 1995; Fletcher et al. 1996). The targeting of Vpr and Vpx to viral particles has been used to incorporate heterologous proteins in virions as either Vpr or Vpx fusion proteins (Wu et al. 1995).

Vpu Protein

The vpu gene is found in HIV-1 and the related chimpanzee lentivirus SIVcpz but not in other retroviruses (Cohen et al. 1988; Myers et al. 1994). It encodes a small integral membrane protein of approximately 80 amino acids that forms an oligomeric complex (Cohen et al. 1988; Malderelli et al. 1993). Expression of the vpu reading frame occurs through a bicistronic message that includes the downstream env reading frame (see Chapter 6. vpu mutants are deficient in viral release and accumulate cell-associated viral proteins (Strebel et al. 1988, 1989; Terwilliger et al. 1989b; Klimkait et al. 1990).

Two distinct activities have been associated with expression of vpu. First, Vpu appears to facilitate the efficient release of Gag proteins from cells (Yao et al. 1992; Geraghty and Panganiban 1993); this activity of HIV-1 Vpu can be detected with both HIV-1 itself and distantly related retroviruses (Göttlinger et al. 1993). This function is replaced by the Env protein for HIV-2, which lacks a vpu gene (Bour and Strebel 1996). The second activity associated with Vpu is the ability to mediate the degradation of CD4. Coexpression of the gp160 precursor and CD4 results in the retention of a complex of these proteins in the ER. The complex is dissociated by Vpu which leads to degradation of the ER-trapped CD4 (Willey et al. 1992a,b). Degradation requires the cytoplasmic domain of CD4 (Chen et al. 1993; Lenburg and Landau 1993; Vincent et al. 1993; Willey et al. 1994).

The two distinct activities of Vpu appear to be regulated by the phosphorylation state of the protein. Phosphorylation of Vpu is absolutely required for CD4 degradation but less important for enhanced virion release (Schubert and Strebel 1994). Retention of Vpu in the ER blocks the Vpu-mediated enhancement of viral release, suggesting that the CD4 degradation activity and the enhancement of viral release occur at distinct locations within the cell (Schubert and Strebel 1994). These two activities of Vpu have central roles in viral assembly by increasing the efficiency of transport of the Env protein through the ER and of release of viral particles. Vpu is able to form a cation-selective ion channel analogous to the influenza A virus M2 protein (Ewart et al. 1996), although the role of this ion channel in mediating Vpu activity is unknown.

Nef Protein

The Nef protein of the primate lentiviruses may have a role in viral assembly. This role is somewhat paradoxical since nef is one of the first genes expressed after HIV-1 infection, along with tat and rev (see Chapter 6. Nef has had several activities attributed to it. Like Vif, Nef affects the capacity of the viral particle to carry out DNA synthesis in a subsequent round of infection, although the mechanism by which Nef exerts this effect is not known (Aiken and Trono 1995; Miller et al. 1995; Schwartz et al. 1995). A role in assembly and release of virus is suggested by the ability of Nef to downregulate surface expression of CD4 (Guy et al. 1987; Garcia and Miller 1991; S. Anderson et al. 1993; Foster et al. 1994; Sanfridson et al. 1994). Down-regulation of CD4 occurs late in its biosynthetic pathway (Aiken et al. 1994; Sanfridson et al. 1994), apparently after surface localization of CD4 (Aiken et al. 1994; Rhee and Marsh 1994). Sequences within the cytoplasmic domain of CD4 are necessary and sufficient for Nef-mediated downregulation (Garcia et al. 1993; Aiken et al. 1994; Anderson et al. 1994), although direct interaction between Nef and these sequences has not been demonstrated. Removal of CD4 from the surface of infected cells can be viewed as a mechanism for reducing potential interactions between Env and CD4 (as seen with Vpu) or alternatively as a strategy for reducing the potential for superinfection or reinfection with newly released virus (Benson et al. 1993). Nef is also found associated with both HIV-1 and HIV-2 particles where a fraction of it is cleaved by the protease (Pandori et al. 1996; Schorr et al. 1996; Welker et al. 1996; Bukovsky et al. 1997). Association with the viral particle requires myristylation and is nonspecific (Bukovsky et al. 1997). The presence of Nef in the viral particle may provide a link with its effects on viral DNA synthesis.