HIV-1 Vif suppresses antiviral immunity by targeting STING

Abstract

HIV-1 infection-induced cGAS-STING-TBK1-IRF3 signaling activates innate immunity to produce type I interferon (IFN). The HIV-1 nonstructural protein viral infectivity factor (Vif) is essential in HIV-1 replication, as it degrades the host restriction factor APOBEC3G. However, whether and how it regulates the host immune response remains to be determined. In this study, we found that Vif inhibited the production of type I IFN to promote immune evasion. HIV-1 infection induced the activation of the host tyrosine kinase FRK, which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN. Mechanistically, the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162. However, the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection. These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING. These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.

Keywords: FRK; Immune evasion; Vif; cGAS–STING.

Fig. 1

Vif suppresses the antiviral immune response. A IFNB mRNA levels in THP-1 cells expressing the HIV-1 regulatory proteins Nef, Rev, Tat, Vif, Vpr, and Vpu and infected with HSV-1 for 12 h (n = 3). B ELISA of IFN-β and TNF in the supernatants of MDMs infected with HIV-1 or HIV-1ΔVif for 24 h (n = 3). C IFNB, CXCL10, ISG15, and TNF mRNA levels in MDMs infected with HIV-1 or HIV-1ΔVif (n = 3). D Tat-Rev mRNA expression in MDMs infected with HIV-1 or HIV-1ΔVif for 24 h (n = 3). E Volcano plots (fold-change vs. t test P value) comparing the gene expression in MDMs infected with HIV-1 or HIV-1ΔVif. F GO enrichment analysis of differentially expressed genes. The data are representative of at least three independent experiments. The data are the means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed unpaired Student’s t test)

Fig. 2

Vif interacts with SHP-1 to inhibit the immune response. A, B Immunoassay of HEK293T cell lysates expressing various vectors and treated with pervanadate. C Direct binding of GST–Vif with His–SHP-1. D The binding of GST–Vif with endogenous SHP-1 from THP-1 cells. E Confocal imaging of HeLa cells transfected with Flag–SHP-1 and infected with HIV-1. F Endogenous interaction of Vif and SHP-1 in HIV-1-infected THP-1 cells. G Immunoassay of lysates from HEK293T cells expressing various vectors. H Impact of SHP-1 shRNA in MDMs. I IFNB, CXCL10, ISG15, and TNF mRNA levels in MDMs transfected with SHP-1 or control shRNA and infected with HIV-1 or HIV-1ΔVif for 24 h (n = 3). The data are representative of at least three independent experiments. The data are the means ± SEMs. ***P < 0.001 (two-tailed unpaired Student’s t test)

Fig. 3

SHP-1 interacts with STING to inhibit the activation of STING. Luciferase assay of IFN-β (A) and ISRE (B) activation in HEK293T cells expressing various vectors (n = 3). C, D Immunoassay of lysates of HEK293T cells expressing various vectors. E Direct binding of GST-STING to His–SHP-1 (left) or to endogenous SHP-1 from peritoneal macrophages (right). F–I Immunoassay of lysates of HEK293T cells expressing various vectors. J Immunoassay of lysates of peritoneal macrophages from WT or me^v/me^v mice infected with HSV-1. The data are representative of at least three independent experiments. The data are the means ± SEMs. **P < 0.01 (two-tailed unpaired Student’s t test)

Fig. 4

SHP-1 inhibits K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162. A, B Immunoassay of HEK293T cell lysates expressing various vectors. C IFNB mRNA levels in STING-deficient Tmem173^−/− MEFs transfected with WT, Y162F, or Y239F mutants of STING together with control or SHP-1 and then infected with HSV-1 for 12 h (n = 3). D Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors (n = 3). E Immunoassay of lysates of transfected HEK293T cells infected with HSV-1. F, G Immunoassay of lysates of HEK293T cells expressing various vectors. H IFNB mRNA levels in Tmem173^−/− MEFs transfected with WT, K150R, K235R, or K337R mutants of STING together with control or SHP-1 and infected with HSV-1 for 12 h (n = 3). I Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors (n = 3). J Immunoassay of lysates from HEK293T cells expressing various vectors. The data are representative of at least three independent experiments. The data are the means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed unpaired Student’s t test)

Fig. 5

Vif facilitates SHP-1-mediated inhibition of STING. A Immunoassay of lysates of HEK293T cells expressing various vectors. B Endogenous interaction of SHP-1 and STING in THP-1 cells infected with HIV-1 or HIV-1ΔVif. C–E Immunoassay of lysates from HEK293T cells expressing various vectors. Luciferase assays of IFN-β (F) and ISRE (G) activation in HEK293T cells expressing the indicated vectors (n = 3). H Immunoassay of lysates of MDMs transfected with SHP-1 or control shRNA and infected with HIV-1 or HIV-1ΔVif. I Immunoassay of lysates of THP-1 cells transfected with SHP-1 or control shRNA and infected with HIV-1 or HIV-1ΔVif. J Immunoblot (native PAGE) demonstrating STING dimerization in THP-1 cells transfected with SHP-1 or control shRNA and infected with HIV-1 or HIV-1ΔVif. K IFNB, CXCL10, ISG15, and TNF mRNA levels in THP-1 cells transfected with the indicated vectors and infected with HSV-1 for 12 h (n = 3). The data are representative of at least three independent experiments. The data are the means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed unpaired Student’s t test)

Fig. 6

ITIM phosphorylation is required for the Vif–SHP-1 interaction and cytokine inhibition. A ITIM mutants of HA–Vif and GST–Vif. B Immunoassay of lysates of HEK293T cells expressing various vectors. C Direct binding of GST–Vif or its ITIM mutant to His–SHP-1 (left) or endogenous SHP-1 from THP-1 cells (right). D IFNB, CXCL10, ISG15, and TNF mRNA levels in MDMs infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F) for 24 h (n = 3). E Immunoblot of lysates of MDMs infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F). F ELISA of IFN-β and TNF in supernatants from (D) (n = 3). G Tat-Rev mRNA expression in MDMs infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F) for 24 h (n = 3). H Immunoblot of lysates of THP-1 cells infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F). I Immunoassay of lysates of THP-1 cells infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F). J Immunoblot (native PAGE) demonstrating STING dimerization in THP-1 cells infected with HIV-1, HIV-1ΔVif, or HIV-1-Vif(Y147F). The data are representative of at least three independent experiments. The data are the means ± SEMs. ***P < 0.001 (two-tailed unpaired Student’s t test)

Fig. 7

FRK phosphorylates the ITIM of Vif to suppress cytokine production. A Immunoassay of lysates of HEK293T cells expressing various vectors. B Immunoassay of lysates of HEK293T cells transfected with the indicated vectors and infected with HIV-1. C Immunoassay of lysates of HEK293T cells. D Immunoblot of the in vitro kinase assay performed with purified GST, GST–Vif(Y147F), or GST–Vif and Myc-FRK. E Immunoassay of lysates of HEK293T cells expressing various vectors. F Impact of FRK shRNA in THP-1 cells. G IFNB, CXCL10, ISG15, and TNF mRNA levels in THP-1 cells transfected with FRK or control shRNA and infected with HIV-1 or HIV-1ΔVif (n = 3). H Immunoblot of lysates from THP-1 cells treated as in G. I Tat-Rev mRNA expression in THP-1 cells transfected with FRK or control shRNA and infected with HIV-1 or HIV-1ΔVif for 24 h (n = 3). J Immunoblot of lysates of THP-1 cells treated with D-65495 or DMSO and infected with HIV-1 or HIV-1ΔVif for the indicated times. K IFNB, CXCL10, ISG15, and TNF mRNA levels in THP-1 cells treated with D-65495 or DMSO and infected with HIV-1 or HIV-1ΔVif (n = 3). The data are representative of at least three independent experiments. The data are the means ± SEMs. **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t test)