HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation

Abstract

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

Extended Data Figure 1. SERINC5 is an inhibitor of HIV-1 infectivity

a, Mapping of the INDELS in the genomic locus spanning SERINC5 exon 2 in JTAg cell clonal populations from Fig. 2a. b, Infectivity of HIV-1 from JTAg cells stably transduced with lentiCRISPR targeting GFP or SERINC5 in three different exons (n = 4, experiment replicated twice). c, Relative expression of SERINC5 in primary cells and in cell lines measured by qPCR normalized by expression of ACTB (n = 3). d, Infectivity of HIV-1 from the indicated cell lines expressing SERINC5 (n = 4, experiments were replicated twice). Mean ± s.d., unpaired two-tailed t-test, ***P < 0.001 e, Expression levels of the five SERINC genes in JTAg cells obtained from RNA-seq.

Extended Data Figure 2. Nef and glycoGag expression result in relocalization of SERINC5 to an endosomal compartment and prevent its incorporation into virions

a, Single round Nef-defective NL4-3 produced by cotransfection of HEK293T cells with plasmids expressing Nef proteins or the empty vector control, and PBJ6-SERINC5–HA: immunoblotting of virions and cell lysates from producer cells. b, Immunofluorescence staining of JTAg cells transfected to express SERINC5–GFP, Nef–HA from HIV-1 isolate 97ZA012 (clade C), from SIV^mac239, HA–glycoGag or an empty vector control. Scale bar, 10 µm.

Extended Data Figure 3. SERINC5 inhibits cytoplasmic delivery of virion content

a, Immunodetection of Cre-recombinase (38 kDa) and p24 in HIV-1 particles. b, Effect of 1 µM AZT or 100 nM T20 on Cre-delivery and virus infectivity (TU, transducing units). c, Immunoblotting of HIV-1 virus particles produced from HEK293T expressing increasing levels of SERINC5–HA. d, Effect of SERINC5 on virus fusion measured with BLAM assay T20 served as a negative control. (n = 4, experiment replicated twice). e, Cre delivery by EBOV-GP pseudotyped HIV-1 particles. f, Inhibition of Cre delivery and counteraction by Nef on HIV-1 from HEK293T expressing SERINC5. Mean ± s.d., n = 4, unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 100 µm.

Extended Data Figure 4. SERINC3 and SERINC5 expression is not induced by interferon nor LPS treatments

a–d, Relative gene expression levels of SERINC3, SERINC5 and CXCL10 in response to treatment with IFN-β and LPS in Jurkat (a), monocyte-derived dendritic cells from two donors (MDDC, b), CD4⁺ primary T cells unstimulated (c) or stimulated with PHA (d) from two donors. Expression of the housekeeping gene OAZ1 was used as a normalization control. Mean ± s.d., n = 3.

Figure 1. Nef counteracts an HIV-1 inhibitor

a, Ratio of the infectivity of NL4-3 and NL4-3Nef⁻ produced from the indicated cell lines and measured on TZM-bl reporter cells. b, The schematic of the heterokaryon assay. c, Infectivity of HIV-1 derived from heterokaryons generated by the indicated cell lines (n = 3, mean ± s.d., unpaired t-test, **P < 0.01.). d, Correlation of SERINC5 expression in producer cells and Nef requirement for infectivity. Colours in a and d represent the same cell lines. Trendline indicates linear regression. (Pearson correlation, two-tailed, P < 0.0001). RPM, reads per million.

Figure 2. SERINC5 and SERINC3 inhibit HIV-1

a, Infectivity of HIV-1 from cells stably transduced with lentiCRISPR (n = 4). gRNA, guide RNA; RT, reverse transcriptase. b, Fluorescence microscopy of reporter cells from a. c, Infectivity of HIV-1 from JTAg SERINC5^−/− and immunoblotting of producer cells. d, Infectivity of HIV-1 from PBMC, co-transfected with CRISPR-Cas9 vectors (n = 3, one experiment performed per donor). e–g, Infectivity of HIV-1 from HEK293T expressing SERINC5–HA. Reporter cells infected with HIV-1 from HEK293T expressing SERINC5–HA(n = 4). h, i, Infectivity of HIV-1 produced from JTAg (h) and JTAg SERINC5^−/− (i) transfected with CRISPR-Cas9 vectors targeting the indicated SERINC genes (n = 4). In i, immunodetection of SERINC3–HA in producer cells. Mean ± s.d., unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 µm.

Figure 3. Determinants of Nef activity against SERINC5 and conservation across different retroviruses

a, The ability of Nef mutants to counteract SERINC5 inhibition of HIV-1 infectivity (n = 4). b, c, Susceptibility of viral pseudotypes to inhibition of infectivity by SERINC5 (n = 4). d, e, Counteraction of SERINC5 by nef alleles and immunoblot from producer cells (d) and glycoGag (e) on HIV-1. f, g, Infectivity of wild-type and glycoGag-defective MLV from HEK293T expressing SERINC5 (n = 4). Mean ± s.d., unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Scale bar, 100 µm.

Figure 4. Nef and glycoGag promote relocalization of SERINC5 to an endosomal compartment and prevent its incorporation into virions

a–e, Immunoblots on viral particles and corresponding cell lysates from nef-defective NL4-3 complemented with plasmids encoding Nef proteins as indicated (a and b), MLV glycoGag (c), VSV-G (d) EBOV-GP (e) and a vector expressing SERINC5–HA. f, Infectivity of HIV-1 from HEK293T cells stably expressing a doxycycline-inducible shRNA targeting AP2 and transfected with PBJ6-SERINC5. Western blot: AP2 in cell lysates derived from producer cells (mean ± s.d., n = 4 unpaired two-tailed t-test, **P < 0.01, experiment replicated twice). g, h, Confocal microscopy of JTAg cells transfected to express SERINC5–GFP with RFP, Nef–RFP (g) or Rab7–RFP (h). Scale bars, 10 µm(g) and 2 µm (h).

Figure 5. SERINC5 inhibits an early step of virus infection

a, Effect of SERINC5 on the generation of HIV-1 late reverse transcription products (n = 3; experiments replicated twice) and the corresponding effect on infectivity. b, Schematic of the nlsCre delivery assay. c, d, Effect of SERINC5 on Cre-delivery by HIV-1 (c) and HIV-1 pseudotyped with VSV-G (d). e, Cre delivery and infectivity by HIV-1-derived from JTAg or JTAg SERINC5−/−. f, Schematic showing the activity of SERINC5 on HIV-1 infectivity and the counteracting mechanism by Nef. Mean ± s.d., n = 4, unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 µm.