UFMylation promotes orthoflavivirus infectious particle production

Abstract

Post-translational modifications play crucial roles in viral infections, yet many potential modifications remain unexplored in orthoflavivirus biology. Here we demonstrate that the UFMylation system, a post-translational modification system that catalyzes the transfer of UFM1 onto proteins, promotes infection by multiple orthoflaviviruses including dengue virus, Zika virus, West Nile virus, and yellow fever virus. We found that depletion of the UFMylation E3 ligase complex proteins UFL1 and UFBP1, as well as other UFMylation machinery components (UBA5, UFC1, and UFM1), significantly reduces infectious virion production for orthoflaviviruses but not the hepacivirus, hepatitis C. Mechanistically, UFMylation does not regulate viral RNA translation or RNA replication but instead affects a later stage of the viral lifecycle. We identified novel interactions between UFL1, and several viral proteins involved in orthoflavivirus virion assembly, including NS2A, NS2B-NS3, and Capsid. These findings establish UFMylation as a previously unrecognized post-translational modification system that promotes orthoflavivirus infection, likely through modulation of viral assembly. This work expands our understanding of the post-translational modifications that control orthoflavivirus infection and identifies new potential therapeutic targets.

Figure 1.. The UFMylation E3 ligase complex proteins promote mosquito-borne orthoflavivirus infection.

(A and B) Focus-forming assay of supernatants from Huh7 cells infected with DENV^NGC or ZIKV^PRVABC59 (48 h, MOI 0.1) after siRNA depletion of the indicated transcripts or non-targeting control (CTRL), shown as % of siCTRL. (C) Cell viability measured after siRNA depletion of the indicated transcripts at 72 hours post-transfection, relative to that of siCTRL, as measured by Cell-Titer GLO assay. (D) Immunoblot analysis of protein expression from Huh7 cells treated with the indicated siRNAs for 72 hours. (E) Focus-forming assay of supernatants harvested from A549 cells infected with ZIKV^PRVABC59 (48 h, MOI 0.1) after siRNA depletion of the indicated transcripts. (F) Immunofluorescence micrographs of Huh7 cells treated with indicated siRNA and then infected with the following viruses for 48 hours (ZIKV^PRVABC59, MOI 0.1; YFV^17D, MOI 0.01; WNV^NY2000, MOI 0.01, or HCV^JFH1, MOI 1), as measured by immunostaining of viral antigen (E for ZIKV, YFV^17D, and WNV, NS5A for HCV; green). Nuclei were stained with Hoechst (blue). Right: Quantification of the percentage of virus-infected Huh7 cells, shown relative to siCTRL. >5000 cells counted for each condition. For all panels, n=3 biologically independent experiments, with bars indicating mean and error bars showing standard error of the mean. *p<0.05, ***p<0.001, ****p<0.0001, or ns, not significant as determined by paired t-test (A and B), one-way ANOVA with Dunnett’s multiple comparisons test (C and E), or two-way ANOVA followed by Šidák’s multiple comparisons test (F).

Figure 2.. The UFMylation E3 ligase complex does not regulate orthoflavivirus translation or RNA replication.

(A) Luciferase activity of Gaussia luciferase-encoding ZIKV^MR766 (ZIKV-GLuc, MOI 0.1) from infected Huh7 cells treated with DMSO, MK0608, or cycloheximide during infection and harvested at the indicated time points. (B) Normalized expression of ZIKV-GLuc from infected Huh7 cells treated with non-targeting control (CTRL) or UFL1 siRNA harvested at the indicated timepoints. (C) Luciferase activity of Gaussia luciferase-encoding ZIKV^MR766 (ZIKV-GLuc, MOI 0.1) from supernatant of infected Huh7 cells treated with DMSO or MK0608 harvested at 24, 48, or 72 hpi. (D) Normalized luciferase activity of ZIKV-GLuc from supernatant of infected Huh7 cells treated with CTRL or UFL1 siRNA and harvested at 24, 48, or 72 hpi.(E) Immunofluorescence micrographs of Huh7 cells treated with the indicated siRNA and then infected with ZIKV^PRVABC59 (36 h, MOI 1) that were immunostained with anti-calnexin (green) and anti-J2 (red) for dsRNA, with the nuclei stained with Hoechst (blue). Scale bar, 10 μm. (F) Normalized luciferase expression of lysates from expression of Huh7 cells transfected with the indicated siRNA and electroporated with a DENV¹⁶⁶⁸¹ subgenomic RNA replicon expressing Renilla luciferase harvested at the indicated timepoints. Treatment with MK0608 was as in (A). For all panels, n=3 biologically independent experiments, with bars indicating mean and error bars showing standard error of the mean. **p<0.01, or ns, not significant, determined by two-way ANOVA with Dunnett’s multiple comparisons test (B, D, and F)

Figure 3.. The UFMylation machinery promotes orthoflavivirus infection.

(A) Immunoblot analysis of Huh7 cells after siRNA depletion of the indicated transcripts or non-targeting control (CTRL). (B) Cell viability measured after siRNA depletion of the indicated transcripts at 72 hours post-transfection, as measured by Cell-Titer GLO assay, relative to the viability of siCTRL. (C-D) Focus-forming assay of supernatants harvested from Huh7 cells infected with DENV^NGC or ZIKV^PRVABC59 (48 h, MOI 0.1) after siRNA depletion of the indicated transcripts, shown as % of siCTRL. (E-F) Focus-forming assay of supernatants harvested from Huh7-UFM1 KO cells transduced with Flag-UFM1^WT or Flag-UFM1^ΔC3 and infected with either DENV^NGC (72 h, MOI 0.1) or ZIKV ^PRVABC59 (48 h, MOI 0.1), shown as % of Flag-UFM1^WT. Immunoblots indicate UFM1-conjugated proteins as those that are higher molecular weight from unconjugated UFM1 but are detected with the anti-UFM1 antibody. For all panels, n=3 biologically independent experiments, with bars indicating mean and error bars showing standard error of the mean. *p<0.05, ** p<0.001, ***p<0.001, ****p<0.0001, or ns, not significant, determined by one-way ANOVA with Dunnett’s multiple comparisons test (B, C, and D) or paired t-test (E and F).

Figure 4.. UFL1 interacts with several DENV and ZIKV proteins.

(A) Schematic of DENV polyprotein, showing membrane topology of viral proteins. (B-C) Immunoblot analysis of anti-V5 immunoprecipitated extracts and inputs, lysed in NP40 buffer (B) or TX-100-RIPA buffer (C), from Huh7 cells stably expressing Flag-UFL1 transfected with plasmids expressing V5-tagged DENV¹⁶⁶⁸¹ proteins. (D-E) Immunoblot analysis of anti-Flag immunoprecipitated extracts and inputs from DENV^NGC-infected or ZIKV^PRVABC59-infected (48 h, MOI 1) Huh7 cells stably expressing Flag-UFL1 or Vector. (F) Immunoblot analysis of anti-Flag immunoprecipitated extracts and inputs from Huh7 cells transfected with DNA plasmids encoding the plasmid-launched ZIKV^MR766-WT or pZIKV ^{MR766-Flag-NS2A} and harvested at 72 hpi. Representative immunoblots from n=3 biologically independent experiments are shown.