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

Abstract

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 (SIVmac239nefAV), 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. SIVmac239nefAV downmodulates CD3, CD4, MHC I and simian tetherin, but cannot counteract SERINC5. In primary rhesus macaque CD4+ T cells, SIVmac239nefAV 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.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4

“…SERINC3 and SERINC5 have also been shown to inhibit HIV-1 by promoting innate immune signaling ( 27 ). Consistent with these observations, the selective disruption of the SERINC5 antagonism of a simian immunodeficiency virus (SIV) Nef protein impaired virus replication in primary CD4 + T cells, attesting to the biological relevance of this function of Nef ( 28 ).…”

Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses

“…We previously defined how these two isolates can robustly downregulate cell-surface CD4, yet only isolate 2410 Nef additionally downregulates SERINC5 from the cell surface efficiently ( Mumby et al 2021 ). The functional ability of the full-length Nef protein derived from SIVmac239 was also included in these experiments, as this SIV Nef protein can downregulate both SERINC5 ( Janaka et al 2021 ) and CD4 from the cell surface efficiently ( Manrique et al 2017 ; Janaka et al 2021 ). Indeed, upon comparing to the eGFP-only negative control, the ancestral PLV Nef protein demonstrated an approximate 2.5-fold significant increase in cell-surface SERINC5 downregulation ( ; fig.…”

Beyond Impairment of Virion Infectivity: New Activities of the Anti-HIV Host Cell Factor SERINC5

“…Maybe the most striking evidence for the functional relevance of the S5 restriction of virus spread in vivo comes from the MLV mouse system, where the knock-out of the S5 gene overcomes the pronounced replication deficit of MLV variants that lack the S5 antagonist GlycoGag [ 36 ]. In line with such a role for S5 in controlling viral spread in vivo, the ability of SIV Nef variants engineered to lack S5 antagonism to boost SIV replication in vivo is significantly reduced [ 37 ]. Several observations from patient cohorts and different natural hosts of lentivirus infection also suggest a crucial role for S5: the magnitude by which lentiviral Nefs can antagonize S5 correlates with the prevalence of the corresponding lentiviral lineage [ 19 ]; correlations were reported for the extent of S5 antagonism with clinical progression and viral load [ 38 , 39 , 40 , 41 ], and levels of SERINC mRNA have been found to be decreased in HIV patients [ 42 ].…”

“…Moreover, a stretch of amino acids in the flexible N-terminal anchor domain of Nef (the S5-antagonism motif, S5AM) has been identified as an additional amino acid motif in Nef that contributes to S5 antagonism but still awaits the identification of the responsible host cell ligands. The S5AM is located in proximity to the binding site of the Nef-associated kinase complex [ 79 ] but mediates distinct yet unknown protein interactions [ 80 ], and the analogous motif in simian immunodeficiency virus (SIV) Nef is important for promoting virus replication in primary CD4 T cells [ 37 ]. In addition, an analysis of patient-derived HIV-1 Nef variants indicated roles for residues K94 and H116 in S5 antagonism [ 40 ].…”