An N-Glycosylated Form of SERINC5 Is Specifically Incorporated into HIV-1 Virions

Abstract

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 , 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. 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 is required neither for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

A Conserved Acidic-Cluster Motif in SERINC5 Confers Partial Resistance to Antagonism by HIV-1 Nef

“…2B and C, which show representative results of an experiment in which Jurkat cells were used to produce virions and quantitative data from three experiments). We confirmed that a 55-kDa form of SERINC5, while the minority species in cells, is the predominant form in virions, an effect due to the selective incorporation into virions of a form of the protein modified by complex glycans (58). The quantitative data suggested that the ΔEDTEE mutant was slightly less efficiently incorporated into virions than wild-type SERINC5 in the absence of Nef; the data also indicated that the ΔEDTEE mutant was excluded from virions by Nef about 2-fold more efficiently than wild-type SERINC5 ( Fig.…”

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

“…4g-i). The disulphide bonds and the location of ECL4, the equivalent of which harbours glycosylated Asn294 in SERINC5 22 , confirms the assigned orientation within the plasma membrane, with both the N-and C-termini of the protein residing in the cytoplasm (Fig. 1b, Supplementary Table 1 and Extended Data Fig.…”

“…Arrowheads and asterisks indicate migration position of glycosylated and non-glycosylated SERINC5, respectively. Note the selective incorporation of the glycosylated form into viral particles 22 d, Class 1 and 2 residues mapped onto a model of SERINC5, in red and blue, respectively. e, Neutralisation of Nef-deficient HIV-1 NL4-3 carrying the JRFL envelope by 2F5 and 4E10 monoclonal antibodies.…”

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

“…Additionally, we noticed that in cells in which Gpr176 was overexpressed, a minor fraction of the WT proteins migrated at approximately 50 kDa ( Fig. 2B; open arrowhead), which possibly reflects the presence of immature or less glycosylated proteins, as reported for other GPCRs and non-GPCR glycoproteins that have been overexpressed [33][34][35][36] .…”