Selective Disruption of SERINC5 Antagonism by Nef Impairs SIV Replication in Primary CD4⁺ T Cells

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 SIV_mac239 Nef protein, we identified residues that are required to counteract SERINC5. This information was used to engineer an infectious molecular clone of SIV (SIV_mac239nef _AV), 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. SIV_mac239nef _AV downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4⁺ T cells, SIV_mac239nef _AV exhibits impaired infectivity and replication compared to wild-type SIV_mac239. These results demonstrate that SERINC5 antagonism can be separated from other Nef functions and reveal the impact of SERINC5 on lentiviral replication.Importance: 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.