Janaka Sanath Kumar, Palumbo Alexandra V, Tavakoli-Tameh Aidin, Evans David T
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison.
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison
J Virol. 2021 Mar 25;95(8). doi: 10.1128/JVI.01911-20. Epub 2021 Jan 27.
The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of the multipass transmembrane protein serine incorporator 5 (SERINC5), and to a lesser extent SERINC3, into virions. In addition to counteracting SERINCs, SIV Nef also downmodulates several transmembrane proteins from the surface of virus-infected cells, including simian tetherin, CD4 and MHC class I (MHC I) molecules. From a systematic analysis of alanine substitutions throughout the SIV239 Nef protein, we identified residues that are required to counteract SERINC5. This information was used to engineer an infectious molecular clone of SIV (SIV239 ), which differs by two amino acids in the N-terminal domain of Nef that make the virus sensitive to SERINC5 while retaining other activities of Nef. SIV239 downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4 T cells, SIV239 exhibits impaired infectivity and replication compared to wild-type SIV239. These results demonstrate that SERINC5 antagonism can be separated from other Nef functions and reveal the impact of SERINC5 on lentiviral replication.: SERINC5, a multipass transmembrane protein, is incorporated into retroviral particles during assembly. This leads to a reduction of particle infectivity by inhibiting virus fusion with the target cell membrane. The Nef proteins of HIV-1 and SIV enhance viral infectivity by preventing the incorporation of SERINC5 into virions. However, the relevance of this restriction factor in viral replication has not been elucidated. Here we report a systematic mapping of Nef residues required for SERINC5 antagonism. Counter screens for three other functions of Nef helped identify two residues in the N-terminal domain of Nef, which when mutated make Nef selectively susceptible to SERINC5. Since Nef is multi-functional, genetic separation of SERINC5 antagonism from its other functions affords comparison of the replication of isogenic viruses that are or are not sensitive to SERINC5. Such a strategy revealed the impact of SERINC5 on SIV replication in primary rhesus macaque CD4 T-cells.
HIV-1和SIV的Nef蛋白通过阻止多跨膜蛋白丝氨酸整合酶5（SERINC5）以及在较小程度上阻止SERINC3整合到病毒粒子中，增强病毒的感染性。除了对抗SERINC蛋白外，SIV Nef还能下调病毒感染细胞表面的几种跨膜蛋白，包括猿猴限制素、CD4和MHC I类（MHC I）分子。通过对SIV239 Nef蛋白中丙氨酸取代的系统分析，我们确定了对抗SERINC5所需的残基。这些信息被用于构建一种SIV的感染性分子克隆（SIV239），该克隆在Nef的N端结构域中有两个氨基酸不同，这使得病毒对SERINC5敏感，同时保留了Nef的其他活性。SIV239能下调CD3、CD4、MHC I和猿猴限制素，但不能对抗SERINC5。在原代恒河猴CD4 T细胞中，与野生型SIV239相比，SIV239的感染性和复制能力受损。这些结果表明，SERINC5拮抗作用可以与Nef的其他功能分离，并揭示了SERINC5对慢病毒复制的影响。：SERINC5是一种多跨膜蛋白，在组装过程中被整合到逆转录病毒颗粒中。这通过抑制病毒与靶细胞膜的融合导致颗粒感染性降低。HIV-1和SIV的Nef蛋白通过阻止SERINC5整合到病毒粒子中增强病毒感染性。然而，这种限制因子在病毒复制中的相关性尚未阐明。在此我们报告了SERINC5拮抗作用所需的Nef残基的系统定位。对Nef的其他三种功能进行反向筛选有助于确定Nef N端结构域中的两个残基，这两个残基突变后会使Nef对SERINC5选择性敏感。由于Nef具有多种功能，将SERINC5拮抗作用与其其他功能进行遗传分离，能够比较对SERINC5敏感或不敏感的同基因病毒的复制情况。这样一种策略揭示了SERINC5对原代恒河猴CD4 T细胞中SIV复制的影响。
