The conserved poxvirus membrane entry-fusion apparatus component OPG147 targets MITA/STING for immune evasion

Abstract

Monkeypox virus (MPXV) causes severe diseases in immunocompromised individuals. How MPXV evades the host defense remain enigmatic. We performed expression screens and identified MPXV OPG147, a membrane fusion machinery protein, as an inhibitor of cGAS-MITA/STING-mediated innate immunity. OPG147 from other poxviruses including the prototypic vaccinia virus (VACV) shows similar functions. OPG147 is associated with MITA/STING and STIM1, a calcium sensor that retains MITA/STING in the ER. OPG147 does not block cGAMP binding to MITA, but inhibits its ISGylation, dimerization/oligomerization and trafficking, thereby suppressing its activation. Mutation of VACV OPG147 F55/T116/T117 to alanine (VACVOPG147/3A) has no effects on its infection and replication, but induces higher innate immune response compared with wild-type VACV in cells and mice. VACVOPG147/3A infection also results in lower viral loads and decreased disease severity in mice. Our findings suggest that OPG147 contributes to immune evasion and is a virulence factor of poxviruses.

Fig 1. Identification of OPG147 as a conserved inhibitor of the cGAS-mediated pathway.

(A) Screening of MPXV-encoded proteins that regulate the cGAS-mediated pathway. HEK293 cells (5 × 10⁴) were transfected with ISRE reporter plasmid (25 ng), expression plasmids for FLAG-cGAS (10 ng), HA-MITA (5 ng) and MPXV-encoded proteins (25 ng) or empty vector for 18 hours before luciferase assays. Data were normalized to empty vector-transfected control. (B) OPG147 inhibits cGAS-MITA-mediated ISRE activation in a dose-dependent manner. HEK293 cells (1 × 10⁵) were transfected with ISRE reporter plasmid (50 ng), expression plasmids for FLAG-cGAS (20 ng), HA-MITA (10 ng) and mOPG147-FLAG (0, 50, 100 ng) or empty vector for 18 hours before luciferase assays and immunoblotting analysis. Empty vector was added to ensure that each transfection receives the same amount of total DNA. (C) OPG147 is a conserved inhibitor of the cGAS-MITA pathway. Sequence identity of OPG147 among orthopoxviruses is shown (Left). HEK293 cells (1 × 10⁵) were transfected with ISRE reporter plasmid (50 ng), expression plasmids for FLAG-cGAS (20 ng), HA-MITA (10 ng), and FLAG-tagged OPG147 from different poxvirus species (50 ng) or empty vector for 18 hours before luciferase assays and immunoblotting analysis (Right). Empty vector was added to ensure that each transfection receives the same amount of total DNA. (D) Effects of OPG147 on transcription of downstream genes induced by VACV. The control THP-1 cells and THP-1 cells stably expressing mOPG147-FLAG or vOPG147-FLAG (1 × 10⁶) were left uninfected or infected with VACV (MOI = 1) for the indicated times before qPCR analysis. (E) Effects of OPG147 on phosphorylation of TBK1, MITA and IRF3 induced by VACV. The control THP-1 cells and THP-1 cells stably expressing mOPG147-FLAG or vOPG147-FLAG (1 × 10⁶) were left uninfected or infected with VACV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (F) Effects of OPG147 on activation of ISRE induced by SeV. HEK293 cells (1 × 10⁵) were transfected with ISRE reporter plasmid (50 ng) and expression plasmids for mOPG147-FLAG or vOPG147-FLAG (0, 50, 100 ng) for 18 hours. The cells were then left untreated or treated with SeV (MOI = 1) for 12 hours before reporter assays and immunoblotting analysis with the indicated antibodies. Empty vector was added to ensure that each transfection receives the same amount of total DNA. Data shown in (B), (C), (D)&(F) are represented as mean ± SEM, n = 3 independent samples. These experiments were repeated for at least two times with similar results. * P < 0.05, ** P < 0.01.

Fig 2. OPG147-deficiency promotes innate immune response to MPXV.

(A) Effects of OPG147-deficiency on phosphorylation of TBK1, MITA and IRF3 induced by MPXV. The control and OPG147 gRNA-edited THP-1 cells (1 × 10⁶) were left untreated or infected with MPXV (MOI = 0.1) for the indicated times before immunoblotting analysis with the indicated antibodies. (B) Effects of OPG147-deficiency on transcription of downstream genes induced by MPXV. The control and OPG147 gRNA-edited THP-1 cells (1 × 10⁶) were left uninfected or infected with MPXV (MOI = 0.1) for the indicated times before qPCR analysis. (C&D) Effects of OPG147-deficiency on production of progeny viruses and cytopathic effects induced by these viruses. The control and OPG147 gRNA-edited THP-1 cells (1 × 10⁶) were infected with MPXV (MOI = 0.1) for 2 hours. The cells were then washed twice with PBS, cultured in RPMI 1640 medium containing 2% FBS for 48 hours. Cells were then centrifuged, and the cell culture medium was collected. Titers of progeny viruses in the cell culture medium were quantified by qPCR (C) and plaque assays (D). (E) Effects of expression of the OPG147 gRNA on transcription of downstream genes induced by HSV-1. The control and OPG147 gRNA-edited THP-1 cells (1 × 10⁶) were left uninfected or infected with HSV-1 (MOI = 1) for the indicated times before qPCR analysis. Data shown in (B), (C), (D)&(E) are represented as mean ± SEM, n = 3 independent samples. All the experiments were repeated for at least two times with similar results. n.s., not significant. * P < 0.05, ** P < 0.01.

Fig 3. OPG147 is associated with MITA.

(A) Effects of OPG147 on transcription of downstream genes induced by cGAMP. The control THP-1 cells and THP-1 cells stably expressing vOPG147 (1 × 10⁶) were left untreated or treated with 2′3′-cGAMP (100 ng/mL) for the indicated times before qPCR analysis. Data are represented as mean ± SEM, n = 3 independent samples. n.s., not significant. *P < 0.05; **P < 0.01. (B) mOPG147 is associated with MITA. HEK293 cells (5 × 10⁶) were transfected with the indicated plasmids. Eighteen hours post-transfection, co-immunoprecipitation was performed with control mouse IgG or anti-HA. The immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies. (C) mOPG147 is colocalized with MITA in the ER. Confocal microscopy of HT1080 cells transfected with ER-GFP, mOPG147-cherry and MITA-FLAG for 24 hours.(D) Viral OPG147 is associated with endogenous MITA following MPXV infection. THP-1 cells (3 × 10⁷) were infected with MPXV (MOI = 0.1) for the indicated times before co-immunoprecipitation and immunoblotting analysis. (E) Key interactions between MITA (light grey) and mOPG147 (dark grey). The inset highlights key residues involved in the interaction: Q315 of MITA corresponds to F55 of mOPG147, while K289 of MITA corresponds to T114 and T115 of mOPG147. (F-G) Association between mOPG147 or its mutants and MITA or its mutants. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids for 18 hours before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Fig 4. OPG147 inhibits ISGylation, dimerization and oligomerization of MITA.

(A) OPG147 has no effects on binding of cGAMP to MITA. The control THP-1 cells and THP-1 cells stably expressing mOPG147-FLAG (3 × 10⁷) were lysed in lysis buffer, followed by co-immunoprecipitation with anti-MITA and Protein G Sepharose. The beads were incubated with 2′3′-cGAMP (1 μg/mL) in binding buffer, washed, and heated to remove denatured proteins. The heat-resistant supernatant was assayed for cGAMP levels by ELISA (histograph). A small aliquot of immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies (lower blots). Data are represented as ± SEM, n = 3 independent samples. n.s., not significant. (B) Binding of mOPG147 to MITA blocks ISGylation of MITA at K289. The image shows a structural model of a mOPG147-MITA complex, where mOPG147 (dark grey) interacts with the cGAMP (orange)-bound MITA homodimer (white). (C) Effects of OPG147 and its mutants on ISGylation of MITA. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids for 18 hours. The cells were then left untreated or treated with 2′3′-cGAMP (1 μM) for 30 min, and further incubated in medium for 1 hour before co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. (D) Effects of OPG147 and its mutants on dimerization and oligomerization of MITA. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids for 18 hours. The cells were then left untreated or treated with 2′3′-cGAMP (1 μM) for 30 min, and further incubated in medium for 1 hour before immunoblotting analysis were performed with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Fig 5. OPG147 blocks cGAMP-triggered dissociation of MITA from STIM1 and trafficking.

(A) Effects of mOPG147 and its mutants on association of MITA with STIM1. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids for 18 hours. The cells were then left untreated or treated with 2′3′-cGAMP (1 μM) for 30 min, and further incubated in medium for 1 hour before co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. (B&C) mOPG147 impairs MITA trafficking. Mita^-/- MLFs reconstituted with MITA-FLAG, co-transfected with RAB9-GFP (for ER) or GM130-GFP (for Golgi) and mOPG147^WT-Cherry or mOPG147^F55A-Cherry, were stimulated with 2′3′-cGAMP (100 ng/mL) for 2 hours before confocal microscopy. Colocalization of RAB9-GFP or GM130-GFP with MITA under each condition was analyzed by calculation of Pearson’s correlation coefficients. Data show mean ± SEM of 3 cells per group (C) and one representative experiment (B). * P < 0.05, ** P < 0.01. (D) mOPG147 impairs recruitment of TBK1 and IRF3 to MITA. HEK293 cells (5 × 10⁶) were transfected with the indicated plasmids. Eighteen hours post-transfection, co-immunoprecipitation was performed with control mouse IgG or anti-HA. The immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies.

Fig 6. OPG147 contributes to VACV evasion of host defense and is a virulence factor in vivo.

(A) Transcription of antiviral genes induced by wild-type VACV and VACV^OPG147/3A. THP-1 cells (1 × 10⁶) were left uninfected or infected with wild-type VACV or VACV^OPG147/3A (MOI = 1) for the indicated times before qPCR analysis. (B) Phosphorylation of TBK1, MITA and IRF3 induced by wild-type VACV and VACV^OPG147/3A. THP-1 cells (1 × 10⁶) were left uninfected or infected with wild-type VACV or VACV^OPG147/3A (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (C) The control vector- and vOPG147-FLAG-transduced THP1 cells (1 × 10⁶) were left un-infected or infected with wild-type VACV or VACV^OPG147/3A (MOI = 1) for the indicated times before qPCR analysis. (D&E) Replication and production of progeny viruses following infection of wild-type VACV and VACV^OPG147/3A. THP-1 cells (1 × 10⁶) were infected with wild-type VACV or VACV^OPG147/3A (MOI = 0.1) for 2 hours. The cells were then washed twice with PBS, cultured in RPMI 1640 medium containing 2% FBS for 48 hours. The cells were centrifuged, and the cell culture medium was collected. Titers of progeny viruses in the cell culture medium were quantified by qPCR (D) and plaque assays (E). (F) ELISA analysis of IFN-β levels in BALF collected from C57BL/6J mice (n = 5 in each group) at 4 days post-infection with wild-type VACV or VACV^OPG147/3A at a dose of 2 × 10⁶ pfu per mouse. (G) Pathological analysis of mouse lungs and spleens. Lungs and spleens were harvested from C57BL/6J mice at 7 days post-infection with wild-type VACV or VACV^OPG147/3A at a dose of 2 × 10⁶ pfu per mouse for hematoxylin-eosin (HE) staining. Scale bars, 400 μm. (H) Measurement of viral loads in mouse lungs and spleens. VACV genomic copy numbers in lungs and spleens of mice infected with wild-type VACV or VACV^OPG147/3A (2 × 10⁶ pfu per mouse) were quantified by qPCR. (I) Survival of C57BL/6J mice (n = 10 in each group) infected with wild-type VACV or VACV^OPG147/3A (2 × 10⁶ pfu per mouse) was monitored daily for 21 days. Kaplan-Meier analysis was used to generate survival curves, and a log-rank test was used to compare groups. Data shown in (A), (C), (D), (E)&(H) are represented as mean ± SEM, n = 3 independent samples. All the experiments were repeated at least two times with similar results. n.s., not significant. n.d., not detectable. d.p.i., days post infiction. * P < 0.05, ** P < 0.01.

Fig 7. A model on OPG147-mediated evasion of the cGAS-MITA/STING pathway.

See text for details.