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 SIVmac239 Nef protein, we identified residues that are required to counteract SERINC5. This information was used to engineer an infectious molecular clone of SIV (SIVmac239nef 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. SIVmac239nef AV downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4+ T cells, SIVmac239nef AV exhibits impaired infectivity and replication compared to wild-type SIVmac239. 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.