SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was required neither for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady-state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild-type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive activity or Nef sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that nonglycosylated SERINC5 is likely subjected to a quality control mechanism that induces its proteasomal degradation.IMPORTANCE SERINC5 is a member of a family of multipass transmembrane proteins that inhibit the infectivity of retroviruses, including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady-state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu β-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host's attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.