Hepatitis C virus core protein blocks interferon signaling by interaction with the STAT1 SH2 domain

Abstract

Emerging data have indicated that hepatitis C virus (HCV) subverts the host antiviral response to ensure its persistence. We previously demonstrated that HCV protein expression suppresses type I interferon (IFN) signaling by leading to the reduction of phosphorylated STAT1 (P-STAT1). We also demonstrated that HCV core protein directly bound to STAT1. However, the detailed mechanisms by which HCV core protein impacts IFN signaling components have not been fully clarified. In this report, we show that the STAT1 interaction domain resides in the N-terminal portion of HCV core (amino acids [aa] 1 to 23). This domain is also required to produce P-STAT1 reduction and inhibit IFN signaling transduction. Conversely, the C-terminal region of STAT1, specifically the SH2 domain (aa 577 to 684), is required for the interaction of HCV core with STAT1. The STAT1 SH2 domain is critical for STAT1 hetero- or homodimerization. We propose a model by which the binding of HCV core to STAT1 results in decreased P-STAT, blocked STAT1 heterodimerization to STAT2, and, therefore, reduced IFN-stimulated gene factor-3 binding to DNA and disrupted IFN-stimulated gene transcription.

FIG. 1.

Full-length HCV core and N-terminal HCV core constructs produce similar levels of P-STAT1 loss and IFN signaling inhibition in Huh7 cells. (A) Map of full-length HCV core and C-terminal truncated or N-terminally truncated HCV core constructs. pC191 is a full-length HCV core construct (aa 1 to 191). pC152 (aa 1 to 152), pC123 (aa 1 to 123), and pC111 (aa 1 to 111) are C-terminally truncated HCV core constructs. pF24-191(aa 24 to 191), pF38-191 (aa 58 to 191), and pF58-191 (aa 58 to 191) are N-terminally truncated HCV core constructs. (B) Expression of full-length and C-terminally truncated HCV core suppresses IFN signaling in Huh7 cells. The full-length HCV core construct pC191; the C-terminally truncated HCV core construct pC152, pC123, or pC111; the N-terminally truncated HCV core construct pF24-191, pF38-191, or pF58-191; or empty vector pCAG was transfected into Huh7 cells treated with or without IFN-α (1,000 IU/ml). The protein lysates were harvested 12 h posttransfection for anti-STAT1 or Western blotting of anti-P-STAT1. Blotting of anti-actin was done as a protein quantitative control. Transfections with full-length HCV core and C-terminally truncated HCV core protein constructs reduced P-STAT1. In contrast, transfection with N-terminal truncated HCV core protein failed to reduce P-STAT1 accumulation. Panel i shows the C-terminally truncated HCV core. Lanes: 1, pCAG (empty) vector; 2, pC191; 3, pC152; 4, pC123; 5, pC111. Panel ii shows the N-terminally truncated HCV core. Lanes: 6, pCAG (empty) vector; 7, pC191; 8, pF24-191; 9, pF38-191; 10, pF58-191.

FIG. 2.

HCV core expression does not downregulate STAT1 gene expression. (A) Reverse transcription-PCR. (B) Real-time PCR. To monitor whether HCV core has an effect on STAT1 gene expression, STAT1 mRNA levels were measured in Huh7 cells or Huh7 cells transfected with the full-length HCV core construct pC191 or the parental empty vector pCAG. GADPH gene levels were measured as a relative quantitative control. HCV expression had no effect on STAT1 mRNA levels. Error bars indicate standard deviations.

FIG. 3.

STAT1 binds to full-length and C-terminally truncated HCV core in Huh7 cells treated with MG132 in an immunoprecipitation (IP) analysis of STAT1 and by Western blotting of anti-HCV core. Protein lysates from Huh7 cells transfected with the full-length HCV core construct pC191, the C-terminally truncated core constructs (pC152, pC123, and pC111), or N-terminally truncated HCV core constructs (pF24-191, pF38-191, and pF58-191) (with the introduction of proteasome inhibitor MG132 [10 μM for 12 h]) and STAT1-immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-HCV core MAb was used for Western blotting. (A) Transfections of C-terminally truncated HCV core constructs. Full-length HCV core protein and C-terminal truncated HCV core bound to STAT1. Lanes: 1, pCAG; 2, pC191; 3, pC152; 4, pC123; 5, pC111; 6, pCAG; 7, pC191; 8, pC152; 9, pC123; 10, pC111. (B) Transfections of N-terminally truncated HCV core constructs. N-terminally truncated HCV core protein did not bind to STAT1. Lanes: 1, pCAG; 2, pF1-191; 3, pF24-191; 4, pF38-191; 5, pF58-191; 6, pCAG; 7, pF1-191; 8, pF24-191; 9, pF38-191; 10, pF58-191. +, with; −, without.

FIG. 4.

N-terminal HCV core is essential for interaction with STAT1, as shown by the immunoprecipitation of HCV core and Western blotting of anti-STAT1. Protein lysates from Huh7 cells transfected with the full-length HCV core construct pC191, the C-terminally truncated core constructs (pC152, pC123, and pC111), or the N-terminally truncated HCV core construct (pF24-191, pF38-191, and pF58-191) (with or without the introduction of proteasome inhibitor MG132 [10 μM for 12 h]) and HCV core-immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-STAT1 MAb was used for Western blotting. (A) C-terminally truncated HCV core protein bound to STAT1. Lanes: 1, pCAG; 2, pC191; 3, pC152; 4, pC123; 5, pC111; 6, pCAG; 7, pC191; 8, pC152; 9, pC123; 10, pC111. (B) The N-terminally truncated HCV core lost the ability to interact with STAT1. Lanes: 1, pCAG; 2, pF1-191; 3, pF24-191; 4, pF38-191; 5, pF58-191; 6, pCAG; 7, pF1-191; 8, pF24-191; 9, pF38-191; 10, pF58-191. +, with; −, without.

FIG. 5.

N-terminal HCV core (aa 1 to 23) inhibits IFN signaling and reduces P-STAT1. (A) Full-length HCV core and N-terminal HCV core (aa 1 to 23) expression suppresses IFN signaling in Huh7 cells. The full-length HCV core construct pF1-191, the N-terminal HCV core construct pF1-23, the N-terminally truncated HCV core construct pF24-191, or the empty vector pCAG was cotransfected with plasmids pISRE-luc (expressing firefly luciferase) and pRL-TK (expressing Renilla luciferase) into Huh7 cells. ISRE-mediated IFN signaling was monitored by a dual-luciferase reporter assay system at 12 h posttransfection. The transfection of the full-length HCV core construct pF1-191 and the N-terminal HCV core construct pF1-23 produced the inhibition of IFN signaling to comparable degrees. In contrast, the transfection of the N-terminally truncated HCV core construct pF24-191 did not suppress IFN signaling. Error bars indicate standard deviations. (B) N-terminal HCV core (aa 1 to 23) interacts with STAT1 in FLAG immunoprecipitation-anti-STAT1 Western blotting. Protein lysates from Huh7 cells transfected with the full-length HCV core construct pF1-191, the N-terminal HCV core construct pF1-23, the N-terminally truncated HCV core construct pF24-191, or empty vector pCAG and anti-FLAG-immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-STAT1 MAb was used for Western blotting. Full-length HCV core and N-terminal HCV core (aa 1 to 23) protein bound to HCV core. Lanes: 1, pCAG; 2, pF1-191; 3, pF1-23; 4, pF24-191; 5, pCAG; 6, pF1-191; 7, pF1-23; 8, pF24-191. +, with; −, without. (C) N-terminal HCV core produces a reduction of P-STAT1. Panel i shows anti-FLAG Western blots to confirm the production of HCV core proteins. Panel ii shows anti-P-STAT1 Western blots. The full-length HCV core construct pF1-191 and the N-terminal HCV core construct pF1-23 reduced the accumulation of P-STAT1. Lanes: 1, pCAG; 2, pF1-191; 3, pF1-23; 4, pF24-191. +, with; −, without.

FIG. 6.

HCV core interacts with STAT1 at the SH2 domain. (A) Map of STAT1 constructs. (a) Full-length STAT1 (pST1-Full, aa 1 to 750). (b) C-terminally truncated STAT1 construct pST1-C684 (Flag STAT1, aa 1 to 684). (c) C-terminally truncated STAT1 construct pST1-C577 (Flag STAT1, aa 1 to 577). (d) STAT1 point mutant constructs at 428/9 M-Glu, a putative DNA binding site (pST1-428/9 M); 701 M-Tyr, a JAK kinase phosphorylation site (pST1-701 M); 713 M-His, a splice site resulting in STAT1b formation (pST1-713 M); and 727 M-Ser, a MAPK phosphorylation site (pST1-727 M). (B) Transfection of C-terminally truncated STAT1 failed to rescue IFN signaling in U3A cells. IFN treatment (12 h) markedly induced ISRE-directed luciferase activity in U3A cells cotransfected with full-length STA1 (aa 1 to 750). In contrast, cotransfection with C-terminally truncated STAT1 constructs pST1-C684 (aa 1 to 684) and pST1-C577 (aa 1 to 577) failed to rescue the IFN signaling. Error bars indicate standard deviations. (C) HCV core immunoprecipitation (IP) and anti-STAT1 Western blotting. Protein lysates from U3A cells cotransfected with the full-length HCV core construct pC191, full-length STAT1, or truncated STAT1 constructs and anti-HCV core immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-STAT1 IgG was used for Western blotting. With the introduction of proteasome inhibitor MG132 (10 μM for 12 h), the HCV core protein bound to full-length STAT1 and the truncated STAT1 construct pST1-C684 but not the truncated STA1 construct pST1-C577. Lanes: 1, pST1-Full; 2, pST1-C684; 3, pST1-C577; 4, pST1-Full; 5, pST1-C684; 6, pST1-C577. +, with; −, without. (D) Anti-Flag immunoprecipitation and HCV core Western blot. Protein lysates from U3A cells cotransfected with the full-length HCV core construct pC191, full-length STAT1, or truncated STAT1 constructs and anti-Flag immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-HCV core IgG was used for Western blotting. With the introduction of proteasome inhibitor MG132 (10 μM for 12 h), full-length HCV core protein bound to full-length STAT1 and truncated STAT1 construct pST1-C684 but not truncated STA1 construct pST1-C577. Lanes: 1, pST1-Full; 2, pST1-C684; 3, pST1-C577; 4, pST1-Full; 5, pST1-C684; 6, pST1-C577. +, with; −, without.

FIG. 7.

HCV core associates with point mutant STAT1. (A) Full-length STAT1 (pST1-Full) transfection restored IFN-induced ISRE-mediated signaling in U3A cells (STAT1 deficiency). IFN treatment (12 h) markedly induced ISRE-directed luciferase activity in U3A cells cotransfected with pSTAT1-Full and pCAG (empty vector). However, in the presence of HCV core the construct pC191, this IFN-stimulated activity was blocked more than threefold. Transfected STAT1 mutants (pST1-701 M and pST1-713 M) have no abilities to rescue IFN signaling in U3A cells cotransfected with pCAG or the HCV core construct pC191. Error bars indicate standard deviations. (B) STAT1 Western blotting. Point mutant STAT1 constructs were cotransfected with the empty vector pCAG (panel i) or the HCV core construct pC191 (panel ii) into U3A cells. The transfection of HCV core reduced expressions of full-length and point mutant STAT1 in U3A cells. Proteasome inhibitor MG132 preserved the expression of STAT1. Lanes: 1, pST1-Full; 2, pST1-428/9 M; 3, pST1-701 M; 4, pST1-713 M; 5, pST1-727 M; 6, U3A (STAT1 deficient); 7 pST1-Full. (C) STAT1 immunoprecipitation-anti-HCV core Western blot. Protein lysates from U3A cells cotransfected with the full-length HCV core construct pC191 and STAT1 point mutant constructs and STAT1-immunoprecipitated lysates were separated by SDS-PAGE and transferred to PVDF membranes. Anti-HCV core MAb was used for Western blotting. With the introduction of the proteasome inhibitor MG132 (10 μM for 12 h), full-length HCV core protein bound to full-length STAT1 and point mutant STAT1. Lanes: 1, pST1-Full; 2, pST1-428/9 M; 3, pST1-701 M; 4, pST1-713 M; 5, pST1-727 M; 6, U3A (STAT1 deficient); 7, pST1-WT; 8, pST1-428/9 M; 9, pST1-701 M; 10, pST1-713 M; 11, pST1-727 M; 12, U3A (STAT1 deficient).