SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue

Abstract

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

Multifunctional Roles of the N-Terminal Region of HIV-1 SF2 Nef Are Mediated by Three Independent Protein Interaction Sites

“…However, these effects are also counteracted by HIV-1 Vpu and Env or require unphysiologically high levels of CD4 and are thus unlikely to account for the effect of Nef on HIV-1 spread in primary CD4 T cells (76)(77)(78). Moreover, two recent studies suggest that the ability of Nef to counteract the restriction to virion infectivity in a single round of infection by SERINC5 also does not translate into improved virus spread in these cells over multiple rounds of infection (75,79). As recently suggested for the MOLT3 T cell line, the Nef-mediated enhancement of HIV-1 spread in such cultures may thus reflect the antagonism of a yet-to-be-identified host cell restriction factor other than SERINC5, which may be counteracted by Nef via a S5AM-dependent mechanism.…”

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

“…Nef genes were amplified by PCR with flanking primers introducing XbaI and MluI restriction sites for cloning into the bi-cistronic CMV promoter-based pCG IRES eGFP vector as described (54). pCG SMM tetherin and pBJ6 human SERINC5-HA protein expression vectors have been previously described (55,56).…”

“…To examine the expression of SIVsmm Nef proteins, HEK293T cells were transfected in 12-well dishes with 2.5 μg DNA of pCG IRES eGFP vectors expressing AU1-tagged Nefs. Two days post-transfection, cells were lysed and proteins separated, blotted and detected as previously described (56).…”

Gene Therapy Applications of Non-Human Lentiviral Vectors

“…As with all primate lentiviruses, SIV has evolved to antagonize these factors. Studies have demonstrated that SIV Vpx and Vpr could degrade SAMHD1 [ 115 ], while Nef could antagonize tetherin [ 116 ] and SERINC5 [ 117 , 118 ]. Furthermore, it has been demonstrated that only SIV cpz and SIV smm Vif could effectively antagonize human APOBEC3G, highlighting one of the possible reasons why cross-species infection has been only observed with these two strains [ 119 ].…”