Department of Medicine, University of California-San Diego, La Jolla, California, USA
Department of Medicine, University of California-San Diego, La Jolla, California, USA.
J Virol. 2018 Oct 29;92(22). doi: 10.1128/JVI.00753-18. Print 2018 Nov 15.
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.
SERINC5 是一种抑制逆转录病毒感染的抑制剂，它被包括 HIV-1 Nef 在内的病毒蛋白所拮抗。SERINC5 对感染的抑制作用与其在病毒粒子中的掺入有关。Nef 拮抗这种抑制作用，可能是通过将 SERINC5 从质膜上的病毒组装部位去除。在评估 SERINC5 的病毒粒子掺入时，我们观察到相对高分子量形式优先存在于病毒粒子中。我们使用各种糖苷酶来确定与病毒相关的 SERINC5 被 N 连接的复杂糖基化修饰，而细胞中大多数 SERINC5 分子量较低，并且被高甘露糖糖基化修饰。SERINC 家族蛋白的序列比对导致我们在 SERINC5 中鉴定出一个保守的 N-糖基化位点 N294。我们突变了该位点，以评估其对糖基化、SERINC5 的限制性活性以及 SERINC5 对 Nef 拮抗作用的敏感性的影响。我们的结果表明，N294 是 SERINC5 中 N-糖基化的主要位点。尽管 N-糖基化既不是限制性活性所必需的，也不是对 Nef 敏感所必需的，但与野生型蛋白相比，糖基化缺陷的 SERINC5（N294A 突变体）的稳态表达减少。与野生型蛋白相比，用 MG132（蛋白酶体抑制剂）处理细胞而非用巴弗洛霉素 A1（溶酶体抑制剂）处理细胞可以部分恢复该突变体的表达。我们得出结论，尽管不是限制性活性或 Nef 敏感性所必需的，但 N 连接的糖基化对于维持 SERINC5 的稳态表达很重要，并且非糖基化的 SERINC5 可能受到诱导其蛋白酶体降解的质量控制机制的影响。SERINC5 是一类多跨膜蛋白家族的成员，它们抑制包括 HIV-1 在内的逆转录病毒的感染性。这些蛋白被掺入病毒粒子中，并抑制靶细胞的感染，除非被 HIV-1 Nef 等病毒拮抗剂拮抗。这些蛋白与之相关的唯一其他生物学功能是形成含丝氨酸的膜脂质。在这里，我们表明 SERINC5 是一种糖蛋白，并且 N-糖基化对于其稳态表达很重要。在没有 N-糖基化的情况下，SERINC5 容易被蛋白酶体降解。尽管如此，N-糖基化既不是 SERINC5 抑制 HIV-1 感染能力所必需的，也不是其对 Nef 拮抗作用敏感所必需的。
