Selective Disruption of SERINC5 Antagonism by Nef Impairs Simian Immunodeficiency Virus Replication in Primary CD4⁺ T Cells

ABSTRACT

The Nef proteins of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (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 major histocompatibility complex 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 2 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. Counterscreens for three other functions of Nef identified two residues in the N-terminal domain of Nef that when mutated make the virus susceptible to SERINC5. Since Nef is multifunctional, separation of SERINC5 antagonism from its other functions allows comparison of the replication of viruses that are or are not sensitive to SERINC5. These experiments reveal the impact of SERINC5 on SIV replication in primary rhesus macaque CD4⁺ T cells.