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. 2015 Oct 8;526(7572):218-23.
doi: 10.1038/nature15400. Epub 2015 Sep 30.

SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

Yoshiko Usami  1 , Yuanfei Wu  1 , Heinrich G Göttlinger  1
Affiliations

SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

Yoshiko Usami et al. Nature. .

Abstract

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Identification of SERINC3 as a candidate target of Nef and glycoGag
a, Anti-HIV-1 CA immunoblot of Nef+, Nef−, and glycoMA+ HIV-1 virions harvested from the indicated fractions of OptiPrep gradients. b, Proteins identified by mass spectrometry in Nef− but not in Nef+ or glycoMA+ virion lysates. The data are from two independent experiments.
Extended Data Figure 2
Extended Data Figure 2. MLV glycoGag inhibits the incorporation of SERINC3 and SERINC5 into HIV-1 virions
a, b, Western blots showing the effects of WT or mutant glycoMA on the incorporation of SERINC3-HA (a) or SERINC5-HA (b) into Nef− HIV-1 virions. The NL4-3/glycoMA proviral construct expresses untagged glycoMA in cis. In all other cases, HA-tagged (a) or FLAG-tagged (b) glycoMA proteins were expressed in trans. The white bands marked by asterisks are caused by co-migrating HIV-1 Pr55gag. Both experiments were performed twice.
Extended Data Figure 3
Extended Data Figure 3. Nef and glycoGag down-regulate SERINC5 from the cell surface
a, SERINC5 re-localizes from the plasma membrane to perinuclear vesicles in the presence of glycoGag. HeLa or U2-OS cells transiently expressing SERINC5-mCherry alone or together with glycoGag were examined by live-cell fluorescence microscopy. b, Nef and glycoGag both down-regulate SERINC5. JurkatTAg cells transiently expressing SERINC5(iHA), either alone or together with NefSF2 or glycoGag, were surface-stained with anti-HA antibody and analyzed by flow cytometry. Per cent fractions of cells expressing SERINC5(iHA) on the surface are indicated. This experiment was performed twice.
Extended Data Figure 4
Extended Data Figure 4. SERINC mRNA expression levels
a, Expression of SERINC family members in uninfected and HIV-infected Jurkat E6.1 cells. RNA was extracted at the peak of infection with WT (Nef+) or Nef− HIV-1NL43, and gene expression was quantified by RNA-seq as reads per kilobase of coding sequence per million reads (RPKM) (n = 1). The HIV-1 budding factor TSG101 and the housekeeping gene HPRT1 are included for comparison. b, Levels of SERINC3 and SERINC5 mRNA (arbitrary units) in cell lines and primary cells, as measured by qRT-PCR (n = 3). PBMC were left unstimulated or stimulated with 0.5 μg/ml phytohemagglutinin (PHA) and 20 U/ml IL-2 for 2 days. c, SERINC5 mRNA expression is not induced by INF-α. PBMC were left untreated or treated with 1000 U/ml human INF-α 2a (PBL Assay Science) for 14 hrs (n = 2). Bars, mean with s.d. NS, not significant (P > 0.05) two-tailed unpaired t-test.
Extended Data Figure 5
Extended Data Figure 5. Exogenous SERINC5 inhibits the fusion of progeny virions with target cells
TZM-bl or A549/CD4/CXCR4 cells were exposed to equal amounts of virus containing BlaM-Vpr, and fusion was analyzed by measuring the Env-dependent increase in blue fluorescence using multiparameter flow cytometry. Virions were produced in 293T cells transfected with an Env− HIV-1 provirus, a vector expressing EnvHXB2 (Env+) or a frameshift mutant (Env−), a vector expressing BlaM-Vpr, and a vector expressing SERINC5 (1 μg or 100 ng) or an equimolar amount of the empty vector (0.7 μg or 70 ng). The percentage of cells displaying increased blue fluorescence is indicated.
Extended Data Figure 6
Extended Data Figure 6. Exogenous SERINC5 reduces the infectivity of Nef− HIV-1 progeny virions for primary target cells
In two independent experiments, PHA-stimulated PBMC from different donors were infected with equal amounts of single-cycle GFP-HIV-1 virions produced in 293T cells in the absence or presence of exogenous SERINC5. Per cent fractions of infected (GFP-positive) cells are indicated.
Extended Data Figure 7
Extended Data Figure 7. SERINC5 incorporation into HIV-1 virions that differ in Nef responsiveness
Recombinant virions were produced in 293T cells co-transfected with the HXB/Env−/Nef− provirus and vectors expressing the poorly Nef-responsive EnvJRFL or the highly Nef-responsive JR (SF V1/V2) Env chimera, along with a vector expressing SERINC5-HA. Empty pBJ5 vector or a version expressing HA-tagged Nef97ZA012 was also co-transfected. SERINC5-HA in purified virions was detected by Western blotting. This experiment was performed twice.
Extended Data Figure 8
Extended Data Figure 8. Characterization of JurkatTAg KO cells
a, Mutant SERINC3 alleles identified in SERINC3 KO clones. b, Mutant SERINC5 alleles identified in SERINC5 KO and SERINC3/5 double-KO clones. The sgRNA target sites are highlighted, and the predicted Cas9 target sites are indicated by arrowheads. Inserted nucleotides are in red. One of the two mutated SERINC5 alleles in JurkatTAg S3−/− S5−/− (1) cells has an inversion between sgRNA target sites A and B. JurkatTAg S5−/− (2) cells harbor 3 mutated SERINC5 alleles. All mutations cause frameshifts and/or large deletions of coding sequence. No WT alleles were detected in any of the KO clones.
Extended Data Figure 9
Extended Data Figure 9. SERINC3 and SERINC5 expression levels in reconstituted double-KO cells
a, SERINC3 protein levels in parental, double-KO, and reconstituted double-KO JurkatTAg cells were compared by Western blotting. SERINC3 migrated close to a prominent background band that was also recognized by the anti-SERINC3 antibody. b, SERINC5 mRNA levels in parental and reconstituted double-KO JurkatTAg cells were compared by qRT-PCR (n = 3).
Extended Data Figure 10
Extended Data Figure 10. Effects of SERINC knockout and reconstitution on HIV-1 replication
Parental, double-KO, and SERINC3 + SERINC5-reconstituted double-KO CD4high JurkatTAg cells were analyzed by immunoblotting with anti-HIV CA at day 9 and day 11 post infection with equal amounts (2 ng p24/ml) of HIV-1NL43 encoding either WT or disrupted versions of NefNL43 or Nef97ZA012.
Figure 1
Figure 1. Inhibition of incorporation of SERINC proteins into HIV-1 virions by Nef correlates with infectivity enhancement
a, b, Western blots showing the effects of Nef proteins from various HIV-1 clades on the incorporation of SERINC3-HA (S3-HA) (a) or SERINC5-HA (S5-HA) (b) into Nef− HIV-1 virions. The white bands marked by asterisks are caused by co-migrating HIV-1 Pr55gag. The experiment shown in (a) was performed twice. Supplementary Information contains full scans for (a, b). c, Ability of Nef proteins from different HIV-1 clades to enhance HIV-1 infectivity. Env−/Nef− HIV-1HXB2 particles trans-complemented with EnvHXB2 were produced in JurkatTAg cells in the absence or presence of the indicated Nef proteins, and infectivities normalized for p24 antigen were determined using TZM-bl indicator target cells (n =3). Bars, mean with s.d. *P < 0.05, **P < 0.01, NS, not significant (P > 0.05), two-tailed unpaired t-test with Welch’s correction in case of unequal variance (F-test, α = 0.025).
Figure 2
Figure 2. Effects of exogenous SERINCs on HIV-1 infectivity
a, Overexpression of SERINC5 in virus producer cells dramatically reduces Nef− HIV-1 progeny virus single-round infectivity. The effects of exogenous SERINC3 (S3) and SERINC5 (S5) were measured using TZM-bl indicator cells (n = 3). b, Western blots showing that virus production, Gag processing, and gp41 (Env) incorporation were unaffected. c, Nef− HIV-1 progeny virions produced in the presence of exogenous SERINC5 are defective in the synthesis of late RT products (n = 2). d, Effects of exogenous SERINCs on the single-round infectivity of VSV G-pseudotyped Nef− HIV-1 virions measured as in Fig. 2a (n = 3). e, VSV G reduces the association of SERINC5-HA (S5-HA) with Env− HIV-1 virions. The HIV-1 proviral plasmid in lane 1 has a disrupted gag gene. This experiment was repeated twice. Supplementary Information contains full scans for (b, e). f, NefSF2 and glycoGag expressed in trans in virus producer cells counteract the effect of exogenous SERINC5 on Nef− HIV-1 progeny virion infectivity (n = 3). Bars, mean with s.d. *P < 0.05, **P < 0.01, NS, not significant (P > 0.05), two-tailed unpaired t-test with Welch’s correction in case of unequal variance.
Figure 3
Figure 3. Effects of depleting SERINCs in virus producer cells
a, Depletion of HA-tagged SERINCs in JurkatTAg cells by specific siRNAs. b, Single-round infectivities of Nef− HIV-1 virions produced in JurkatTAg cells (n = 3) subjected to non-targeting siRNA or to siRNAs targeting SERINC3 (si_S3), SERINC5 (si_S5), or both (si_S3+5). c, Single-round infectivities of Nef− HIV-1 virions produced in primary MDM subjected to siRNAs (n = 3). d, Simultaneous depletion of SERINC3 and SERINC5 has negligible effects on Nef− HIV-1 progeny virion infectivity when Nef or glycoGag are provided in trans (n = 3). e, The effects of depleting SERINC3 together with SERINC5 on virus infectivity are governed by the same determinants in gp120 V1/V2 that govern Nef-responsiveness (n = 3). f, Western blots showing that the combined siRNAs targeting SERINC3 and SERINC5 did not affect particle production, Gag processing, or Env incorporation. Supplementary Information contains full scans for (a, f). Bars, mean with s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant (P > 0.05) two-tailed unpaired t-test with Welch’s correction in case of unequal variance. The experiments shown in (a) and (b) were performed twice.
Figure 4
Figure 4. Effects of SERINC knockout and reconstitution on HIV-1 infectivity
a, Single-round infectivities of Nef− HIV-1 progeny virions produced in parental or in KO JurkatTAg cells lacking SERINC3, SERINC5, or both (n = 3). b, TZM-bl cells were incubated with equal amounts of single-cycle Nef− HIV-1-GFP produced in parental or double-KO cells lacking SERIN3 and SERINC5. Infected TZM-bl cells expressing GFP were detected by fluorescence microscopy. c, Effects of Nef and glycoGag provided in trans on the single-round infectivities of Nef− HIV-1 virions produced in parental or double-KO cells (n = 3). d, Effects of introducing expression cassettes for SERINC3, SERINC5, or both into the double-KO cells on Nef− HIV-1 progeny virus infectivities (n = 3). Bars, mean with s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant (P > 0.05) two-tailed unpaired t-test with Welch’s correction in case of unequal variance. The experiments shown in (a) and (b) were repeated three times.
Figure 5
Figure 5. Nef counteracts inhibition of HIV-1 replication by SERINC3 and SERINC5
a, Effect of Nef on HIV-1 spreading in Jurkat E6. 1 cells infected at a low input virus concentration (100 pg p24/ml). Gag protein expression in the cultures 9 and 13 days post infection was examined by Western blotting as a measure of virus replication. b, c, Effects of Nef on virus spreading in parental JurkatTAg cells, double-KO cells lacking SERINC3 and SERINC5, and SERINC3 +SERINC5-reconstituted double-KO cells. The spreading of HIV-1NL43-based viruses encoding either WT or disrupted versions of NefNL43 or Nef97ZA012 was examined by Western blotting of cell lysates with anti-CA antibody 9 days post infection with 20 ng p24/ml (b), or by monitoring p24 accumulation in the supernatants after infection with 4 ng p24/ml (c). Relatively high input virus concentrations were used to compensate for low CD4 levels on JurkatTAg cells. The experiment shown in (b) was repeated twice. Supplementary Information contains full scans for (a, b).
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