SERINC5 is the primary target of retroviral antagonists among human SERINC paralogs and requires a tri-cysteine motif for its counteraction by HIV-1 Nef.

Abstract

The human transmembrane protein SERINC5 incorporates into budding HIV-1 particles and inhibits virus fusion with target cells. Although three other human SERINC paralogs display antiviral activity, their contribution to retroviral restriction and their susceptibility to viral antagonists have not been systematically compared. HIV-1 Nef, Moloney murine leukemia virus (MoMLV) glycoGag, and the equine infectious anemia virus (EIAV) S2 glycoprotein independently evolved mechanisms to counteract SERINC5. Here, we show that all human SERINC paralogs are incorporated into HIV-1 virions despite their differing antiviral activities. However, compared with the other SERINC paralogs, SERINC5 is selectively and efficiently targeted by all retroviral antagonists examined, supporting the notion that SERINC5 has exerted the strongest selective pressure on retroviral evolution. We identified a tri-cysteine motif within intracellular loop 4 (ICL4) as a critical determinant of SERINC5 antagonism by Nef. Disruption of this motif impaired Nef-mediated internalization, exclusion from virions, and full rescue of HIV-1 infectivity by diverse HIV-1 and SIV Nef alleles, but had no effect on antagonism by MoMLV glycoGag or EIAV S2, indicating that these viral proteins recognize distinct molecular features of SERINC5. Transfer of ICL4 to SERINC2 was insufficient to confer Nef sensitivity, demonstrating that the tri-cysteine motif is necessary but not sufficient for antagonism. Finally, we show that this motif is palmitoylated, suggesting that palmitoylation contributes to SERINC5 recognition by Nef. Together, these findings establish SERINC5 as the principal target of retroviral antagonists and demonstrate that its recognition by Nef depends on the broader structural context of SERINC5.

Importance: The SERINC family of multipass transmembrane proteins includes host factors capable of restricting retrovirus infectivity. Among the five human paralogs, SERINC5 displays the strongest antiviral activity and is the only member efficiently targeted by all known retroviral antagonists, including HIV-1 Nef, MLV glycoGag, and the EIAV S2. This selectivity indicates that the evolutionary pressure exerted by SERINC proteins on retroviruses has been primarily driven by SERINC5 rather than by its relatives. Through structure-guided mutagenesis, we identified a tri-cysteine motif within intracellular loop 4 as a crucial determinant for SERINC5 downregulation by Nef, providing a molecular explanation for its distinctive susceptibility. These findings establish SERINC5 as the principal target of viral counteraction and suggest that its molecular features have contributed to shaping the adaptation of primate lentiviral Nef proteins to the host.