S2 from equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3

Abstract

The lentivirus equine infectious anemia virus (EIAV) encodes the small protein S2, a pathogenic determinant that is important for virus replication and disease progression in horses. No molecular function had been linked to this accessory protein. We report that S2 can replace the activity of Negative factor (Nef) in HIV-1 infectivity, being required to antagonize the inhibitory activity of Serine incorporator (SERINC) proteins on Nef-defective HIV-1. Like Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor, Adaptor protein 2 (AP2). Accordingly, functional endocytic machinery is essential for S2-mediated infectivity enhancement, and S2-mediated enhancement is impaired by inhibitors of clathrin-mediated endocytosis. In addition to retargeting SERINC5 to a late endosomal compartment, S2 promotes host factor degradation. Emphasizing the similarity with Nef, we show that S2 is myristoylated, and, as is compatible with a crucial role in posttranslational modification, its N-terminal glycine is required for anti-SERINC5 activity. EIAV-derived vectors devoid of S2 are less susceptible than HIV-1 to the inhibitory effect of both human and equine SERINC5. We then identified the envelope glycoprotein of EIAV as a determinant that also modulates retroviral susceptibility to SERINC5, indicating that EIAV has a bimodal ability to counteract the host factor. S2 shares no sequence homology with other retroviral factors known to counteract SERINC5. Like the primate lentivirus Nef and the gammaretrovirus glycoGag, the accessory protein from EIAV is an example of a retroviral virulence determinant that independently evolved SERINC5-antagonizing activity. SERINC5 therefore plays a critical role in the interaction of the host with diverse retrovirus pathogens.

Keywords: restriction factor; retrovirus; virus infection.

Fig. 1.

EIAV S2 and HIV-1 Nef are functionally similar infectivity-promoting factors. (A and B) S2 repairs the defective infectivity of Nef-defective single-cycle HIV-1_NL4-3 produced in JTAg cells. S2 was expressed in place of Nef within the HIV-1_NL4-3 provirus (A) or in trans, fused to an HA tag at the C terminus (B). (C and D) S2 and Nef have the same effects on infectivity because S2 does not affect the infectivity of HIV-1_NL4-3 derived from CEMx174 (C) or of HIV-1_NL4-3 pseudotyped with VSV-G or JR-FL env (D). Relative infectivity is expressed as the percent of the infectivity of WT HIV-1_NL4-3 in the absence of S2 expression. Error bars represent the SD of the mean calculated from quadruplicate determinations. B and D include Western blot detection of S2-HA and β-actin in lysates of virus-producing cells.

Fig. S1.

The addition of an N-terminal HA tag impairs the anti-SERINC5 activity of S2. Virus was produced by transfecting HEK293T cells to express WT or mutant S2-HA and SERINC5. Western blotting shows the expression of HA-tagged S2 proteins and β-actin in lysates of producer cells. Infectivity is expressed as a percentage relative to the infectivity of Nef-defective HIV-1NL4-3 produced in the presence of WT untagged S2. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. S2.

S2 derived from Wyoming and SPEIAV19 EIAV isolates have comparable ability to counteract SERINC5 inhibition of Nef-defective HIV-1_NL4-3 and EIAV. Virus was produced by transfecting HEK293T cells to express the indicated S2 alleles. Infectivity is expressed as a percentage relative to the infectivity of Nef-defective HIV-1_NL4-3 produced in the presence of EIAV_WYOMING S2. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. 2.

S2 counteracts the inhibition of HIV-1 by SERINC5 and SERINC3. (A) The effect of S2 on the infectivity of Nef-defective HIV-1_NL4-3 produced in JTAg cells requires endogenously expressed SERINC5 and SERINC3. Shown is the infectivity of Nef-defective HIV-1^NL4-3 produced in JTAg cells knocked out for SERINC5 or knocked out for both SERINC5 and SERINC3. (B) S2 preserves the infectivity of Nef-defective HIV-1_NL4-3 from inhibition by PBJ6-SERINC5-HA transfected in producer HEK293T cells. Relative infectivity is expressed as the percent of the infectivity of Nef-defective HIV-1_NL4-3 in the absence of S2 expression. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. S3.

S2 counteracts the inhibition of HIV-1 by SERINC3. S2 preserves the infectivity of Nef-defective HIV-1_NL4-3 from inhibition by PBJ6-SERINC3-HA transfected in producer HEK293T cells. Relative infectivity is expressed as a percent of the infectivity of Nef-defective HIV-1_NL4-3 in the absence of S2 expression. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. 3.

S2 prevents virion incorporation of SERINC5 and SERINC3. (A and B) S2 expression in HEK293T cells producing Nef-defective HIV-1_NL4-3 prevents virion incorporation of SERINC5-HA (A) and SERINC3-HA (B). (C and D) Western blotting of virion pellets and lysates of the respective producer cells. S2-HA alters the steady-state expression level of SERINC5-HA (C) but not CXCR4-FLAG (D). Shown are Western blots of lysates from HEK293T cells that were cotransfected with PBJ5 plasmid encoding S2-HA and increasing amounts of SERINC5-HA (C) or CXCR4-FLAG (D) expressed from PCDNA vectors.

Fig. 4.

Counteraction of SERINC5 by S2 requires clathrin-dependent endocytosis and a conserved putative AP2-binding motif. (A and B) S2 causes the relocalization of SERINC5-GFP from the plasma membrane to a Rab7-labeled compartment. Shown is fluorescence confocal imaging of JTAg cells transfected to express the indicated proteins. (C) The ability of S2 expressed in trans to counteract SERINC5 is impaired by the clathrin-dependent endocytosis inhibitors Dyn2K44A and AP180-C in HEK293T cells transfected to produce Nef-defective HIV-1_NL4-3. (D) ClustalW alignment of S2 amino acid sequences derived from four isolates representing the four major EIAV groups: EIAV_wy (WY; GenBank accession no. AAC03764), EIAV_IRE (IRE; GenBank accession no. AFW99166), EIAV_LIA (LIA; GenBank accession no. AAK21109), and EIAV_MIY (MIY; GenBank accession no. AFV61766). Putative functional motifs are boxed and color-coded. (E) The integrity of the ExxxLL sequence is required for S2 to counteract the activity of SERINC5 on Nef-defective HIV-1_NL4-3. Virus was produced by transfecting HEK293T cells expressing WT or mutant S2-HA. Western blotting shows the expression of S2-HA and β-actin in lysates of producer cells. Infectivity in C and E is expressed as a percentage relative to Nef-defective HIV-1_NL4-3 produced in the presence of WT S2-HA and the absence of inhibitors. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. S4.

A putative SH3-binding PxxP motif is not required for S2 to counteract SERINC5 inhibition of the infectivity of Nef-defective HIV-1_NL4-3. Infectivity is expressed as a percentage relative to the infectivity of Nef-defective HIV-1_NL4-3 produced in the presence of WT S2-HA and SERINC5. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. 5.

The role of N-terminal glycine for S2 activity. (A) Gly² is required for SERINC to counteract S2-HA on Nef-defective HIV-1_NL4-3. The virus was produced by transfecting HEK293T cells to express WT or mutant S2-HA. Western blotting shows the expression of S2-HA and β-actin in lysates of producer cells. Infectivity is expressed as the percentage relative to the infectivity of Nef-defective HIV-1_NL4-3 produced in the presence of WT S2-HA. Error bars represent the SD of the mean calculated from quadruplicate determinations. (B) S2 is myristoylated. Shown is Western blotting to detect HA-tagged proteins in the myristic acid–azide–enriched fraction and the corresponding whole lysates. (C) Gly² is required for S2-HA localization at the cell membrane. Shown are immunofluorescence confocal images of JTAg cells transfected to express the indicated proteins.

Fig. 6.

EIAV env confers virus particles with partial resistance to SERINC5. (A) EIAV is less susceptible than HIV-1 to SERINC5 inhibition. Shown is the inhibition of the infectivity of S2-defective EIAV (Left) and Nef-defective HIV-1 (Right) virion particles by SERINC5 and the counteraction by S2. (B and C) The sensitivity of S2-defective EIAV (B) and S2-defective EIAV pseudotyped with MLV-A env (C) to human and equine SERINC5. (D) The susceptibility of HIV-1 particles to SERINC5 when pseudotyped with EIAV- or HIV-1 env in the absence of S2. All panels show the infectivity of single-cycle EIAV and HIV-1 virus particles produced from HEK293T cells transfected to express SERINC5 and S2 as indicated. Infectivity is expressed as the percentage relative to the infectivity of Nef-defective HIV-1_NL4-3 or S2-defective EIAV produced in the presence or absence of SERINC5 overexpression. Error bars represent the SD of the mean calculated from quadruplicate determinations.

Fig. S5.

Alignment of equine and human SERINC5 protein sequences. Sequences corresponding to proteins with GenBank accession numbers XP_001503924 (Equus caballus) and NP_001167543 (Homo sapiens) were aligned using EMBOSS Needle (www.ebi.ac.uk/Tools/psa/emboss_needle/).