Redox cycling of viral RNA polymerase controls picornavirus replication

Figures

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  Fig. 1. MSRB3 plays an important role in FMDV infection. a GeCKO screening for candidate genes. The difference analysis results reflected the relationship between P values and the log fold change of all genes. b PK-15 cells were edited with a control or two individual sgRNAs targeting the MSRB3 gene by the CRISPR/Cas9 method to establish stable cell lines. MSRB3-deficient and control PK-15 cells were seeded into 12-well plates for 48 h before cell viability assays. c–f MSRB3-deficient and control PK-15 cells were infected with FMDV (MOI, 0.2) for the indicated times before qPCR (c) and immunoblot (d) analyses, for 24 h before determination of progeny virus titer in the supernatant by TCID50 assay (e), or for 12 h before flow cytometry analysis (f). g–i PK-15 cells were transduced with vector or MSRB3 expression plasmids by lentivirus-mediated gene transfer to establish stable cell lines. MSRB3-overexpressed and control PK-15 cells were infected with FMDV (MOI, 0.2) for the indicated times before immunoblot analysis (g), for 24 h before determination of progeny virus titer in the supernatant by TCID50 assay (h), or for 12 h before flow cytometry analysis (i). j IBRS-2 and WSL cells were…

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  Fig. 2. MSRB3 promotes FMDV replication by stabilizing viral 3D protein. a Effects of MSRB3 deficiency on FMDV attachment and internalization. b HEK293T cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. VP0 is the precursor protein of VP4 and VP2. c PK-15 cells were infected with FMDV (MOI, 0.5) for the indicated times. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. d The binding affinity of MSRB3 to FMDV-3D was detected by the ForteBio Octet Red system. The vertical and horizontal axes represent the light-shift distance (nm) and association/dissociation times, respectively. e The control (Con) PK-15 cells were transduced with empty vector (Vec), and MSRB3-deficient PK-15 cells were transduced with Vec, MSRB3 or MSRB3-C82A by retrovirus-mediated gene transfer to establish stable cell lines with blasticidin S (10 μg/mL) selection. The indicated cell lines were infected with FMDV (MOI, 0.2) for 16 h before qPCR and TCID50 analyses. The blot shows the expression levels of MSRB3 in the indicated cell lines as detected by anti-MSRB3 antibody. f, h, i HEK293T cells were transfected with the indicated plasmids…

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  Fig. 3. RSAD1 inhibits FMDV replication by catalyzing methionine oxidation of viral 3D protein and promoting its degradation. a MSRB3-deficient and control PK-15 cells were transfected with plasmids expressing GST or GST-FMDV-3D, and FMDV-3D-associated host factors were immunoprecipitated and subjected to silver staining. The differential bands between the two groups were separated for mass spectrometry identification. b RSAD1-overexpressed and control PK-15 cells were infected with FMDV (MOI, 0.2) for the indicated times before qPCR analysis. c RSAD1-deficient and control PK-15 cells were infected with FMDV (MOI, 0.2) for 24 h before the TCID50 assay. d The control (Con) PK-15 cells were transduced with empty vector (Vec), and RSAD1-deficient PK-15 cells were transduced with Vec, RSAD1 or it mutants by retrovirus-mediated gene transfer to establish stable cell lines with blasticidin S (10 μg/mL) selection. The indicated cell lines were infected with FMDV (MOI, 0.2) for 24 h before the TCID50 assay. e, h, j, k, m HEK293T cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. f GST-tagged RSAD1 proteins (2 μg) produced in eukaryotic (Eu) or prokaryotic…

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  Fig. 4. RSAD1 promotes FMDV-3D degradation through the autophagy-lysosome pathway. a Detection of ThT fluorescence signal in the indicated groups. Vec: Vector. b Detection of the aggregation of FMDV-3D and its mutant by gel filtration experiments. c PK-15 cells were edited with a control or two individual sgRNAs targeting the TOLLIP gene by the CRISPR/Cas9 method to establish stable cell lines. TOLLIP-deficient and control (Con) PK-15 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. d Domain architecture of TOLLIP. aa: amino acid. e The Con PK-15 cells were transduced with empty vector (Vec), and TOLLIP-deficient PK-15 cells were transduced with Vec, TOLLIP or its mutants by retrovirus-mediated gene transfer to establish stable cell lines with blasticidin S (10 μg/mL) selection. The corresponding cell lines were transfected with the indicated plasmids for 24 h before immunoblot analysis. f MSRB3-deficient and control PK-15 cells were infected with FMDV (MOI, 0.2) for the indicated times before immunoblot analysis. g, h MSRB3-deficient and control PK-15 cells were infected with FMDV (MOI, 0.2) for 8 h, fixed with 4% paraformaldehyde, and stained with…

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  Fig. 5. RSAD1-mediated methionine oxidation of viral 3D protein impairs FMDV pathogenicity in mice. a The RdRp activity of FMDV-3D and its mutants was determined by RNA elongation assays. b Construction strategy of a full-length FMDV cDNA clone containing M194A and M403A mutations in the 3D fragment. c, d RSAD1-deficient or control PK-15 cells were infected with wild-type rFMDV or rFMDV-mut strains (MOI, 0.1) for the indicated times before one-step growth curve determination (c) and immunoblot analysis (d). e Five-day-old C57BL/6J suckling mice were challenged subcutaneously with rFMDV-WT (7 × 104 PFU per mouse, n = 16) or rFMDV-mut (7 × 104 PFU per mouse, n = 15), and the survival of mice was monitored daily throughout the experiment. f, g Five-day-old C57BL/6J suckling mice were challenged subcutaneously with rFMDV-WT (7 × 104 PFU per mouse, n = 10) or rFMDV-mut (7 × 104 PFU per mouse, n = 13) and then euthanized on day 3 postinfection. The indicated tissues were collected immediately for viral load determination by qPCR (f) and histopathological analysis by H&E staining (g). Scan bars, 50 μm. Graphs in c show mean ± SD (n = 3 biological replicates). Graphs in f show the mean from one…

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  Fig. 6. MSRB3 deficiency attenuates the susceptibility of mice to FMDV, EV71 or EMCV challenge. a–c Eight-week-old sex-matched Msrb3+/+ (n = 10) and Msrb3−/− (n = 12) mice were challenged intraperitoneally with FMDV (2.8 × 106 PFU per mouse), 5-day-old Msrb3+/+ (n = 11) and Msrb3−/− (n = 14) suckling mice were challenged subcutaneously with EV71 (2 × 107 PFU per mouse), and 8-week-old sex-matched Msrb3+/+ (n = 10) and Msrb3−/− (n = 11) mice were challenged intraperitoneally with EMCV (7 × 105 PFU per mouse) followed by daily monitoring. d–i Eight-week-old sex-matched Msrb3+/+ (n = 12) and Msrb3−/− (n = 11) mice were challenged intraperitoneally with FMDV (2.8 × 106 PFU per mouse), 5-day-old Msrb3+/+ (n = 13) and Msrb3−/− (n = 12) suckling mice were challenged subcutaneously with EV71 (2 × 107 PFU per mouse), and 8-week-old sex-matched Msrb3+/+ (n = 8) and Msrb3−/− (n = 11) mice were challenged intraperitoneally with EMCV (7 × 105 PFU per mouse). Mice were euthanized on day 3 postinfection, and the indicated tissues were collected immediately for viral load determination by qPCR (d–f) and histopathological analysis by H&E staining and immunostaining with anti-VP1 antibodies (g–i). Scan…

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  Fig. 7. RSAD1 deficiency potentiates the susceptibility of mice to FMDV, EV71, or EMCV challenge. a–c Eight-week-old sex-matched Rsad1+/+ (n = 10) and Rsad1−/− (n = 10) mice were inoculated intraperitoneally with FMDV (2.8 × 106 PFU per mouse), 5-day-old Rsad1+/+ (n = 16) and Rsad1−/− (n = 13) suckling mice were inoculated subcutaneously with EV71 (2 × 107 PFU per mouse), and 8-week-old sex-matched Rsad1+/+ (n = 11) and Rsad1−/− (n = 10) mice were inoculated intraperitoneally with EMCV (7 × 105 PFU per mouse). The survival of mice was recorded daily throughout the experiment. d−i Eight-week-old sex-matched Rsad1+/+ (n = 11) and Rsad1−/− (n = 10) mice were inoculated intraperitoneally with FMDV (2.8 × 106 PFU per mouse), five-day-old Rsad1+/+ (n = 12) and Rsad1−/− (n = 12) suckling mice were inoculated subcutaneously with EV71 (2 × 107 PFU per mouse), and 8-week-old sex-matched Rsad1+/+ (n = 12) and Rsad1−/− (n = 12) mice were inoculated intraperitoneally with EMCV (7 × 105 PFU per mouse). Mice were necropsied on day 3 postinfection, and the indicated tissues were collected immediately for viral load determination by qPCR (d–f) and histopathological analysis by H&E staining and…