RING finger protein 5 is a key anti-FMDV host factor through inhibition of virion assembly

Figures

• 

  Fig 1. RNF5 interacts with FMDV VP1 protein. (A) Exogenous co-immunoprecipitation (Co-IP) assay in HEK293T cells. HEK293T cells were transfected with HA-VP1 and Myc-RNF5 or vector, and the cell lysates were immunoprecipitated with Myc antibody, followed by immunoblot with Myc and HA antibodies. (B) The schematic presentation of RNF5 truncated mutants. (C) The N-terminal domain of RNF5 is the main domain interaction with VP1. HEK293T cells were transfected with HA-VP1 and Myc-RNF5 or its truncations, and the cell lysates were immunoprecipitated with Myc antibodies, followed by immunoblot with Myc and HA antibodies. (D) Colocalization of VP1 with RNF5 or its truncations. HEK293T cells were transfected with HA-VP1 and Myc-RNF5 or its truncations and were then analyzed using immunofluorescence staining with anti-HA (green), anti-Myc (red), and DAPI (blue) and microscopy (Left). The co-localization analysis was expressed as Pearson’s correlation coefficient, measured for individual cells by Image J (Right). (E) Endogenous RNF5 interacted with VP1, VP2, and VP3 during FMDV infection. IBRS-2 cells were infected with or without FMDV for 12 h, and then cell lysates were immunoprecipitated with…

• 

  Fig 2. RNF5 inhibits FMDV replication. (A-C) FMDV replication was restricted by RNF5 ectopic expression. IBRS-2 cells seeded into 6-well plates were transfected with RNF5 or RNF81 plasmids in a dose-dependent manner for 24 h and then inoculated with FMDV at an MOI of 1 for 8, or 16 h. The infected cells at 8 h were harvested for detection of FMDV mRNA abundance using RT-PCR (A). The infected cells at 16 h were harvested for detection of RNF5 and FMDV VP1 protein expression using immunoblot (B), intracellular and extracellular progeny viral titers by assessing TCID50 (C). (D-F) Down-regulation of RNF5 promotes FMDV replication. IBRS-2 cells seeded into 6-well plates were transfected with 150 nM NC, siRNA-1, or siRNA-2 of RNF5 or RNF81 for 24 h, followed by infection with FMDV (MOI = 1) for 8 h or 16 h. The virus RNA at 8 h was extracted, and RT-PCR experiments were performed (D). The protein expression levels of endogenous RNF5 and viral VP1 proteins at 16 h were detected by immunoblot (E). FMDV yields were determined by TCID50 assay (F). (G) Confirmation of successful knockout of RNF5 in PK-RNF5-KO cell line by immunoblot. (H-J) Knockout of pRNF5 in PK-15 cells promotes FMDV progeny…

• 

  Fig 3. RNF5 degrades FMDV VP1 protein and affects virion assembly. (A) RNF5 induces the reduction of VP1 in a dose-dependent manner. HEK293T cells were transfected with HA-VP1 plasmid and increased quantities of Myc-RNF5 plasmids. The expression of HA-VP1 and Myc-RNF5 was detected by immunoblot. (B) The effect of endogenous RNF5 on VP1 during FMDV infection. PK-RNF5-KO, IBRS-RNF5-KO, or WT cells were infected with FMDV (MOI = 0.5) for 24 h, and then endogenous RNF5 and VP1 were detected using immunoblot. (C) RNF5 regulates the half-life of VP1 protein. HEK293T cells were co-transfected with HA-VP1 and Myc-RNF5 or Myc-RNF5C42S plasmids. After 16 h, the cells were treated with CHX (100 μg/ml) for 0, 6, and 12 h before immunoblot. (D) The mRNA expression level of FMDV was detected by RT-PCR. PK-15 cells were transfected with RNF5 or RNF5C42S plasmids and then infected with FMDV (MOI = 0.5) for 24 h. (E) FMDV titers were determined by TCID50 assay. PK-15 cells were transfected with RNF5 or RNF5C42S plasmids and then infected with FMDV (MOI = 0.5) for 36 h. TCID50 assays were performed in BHK-21 cells. (F) RNF5 promotes proteasomal degradation of VP1. HA-VP1 was transfected with Myc-RNF5 or…

• 

  Fig 4. RNF5 catalyzes the K48-linked polyubiquitination of VP1. (A) RNF5 increased the ubiquitination of VP1. Flag-VP1 and Myc-RNF5 or Myc-RNF5C42S were co-transfected into HEK293T cells with HA-Ub, and then treated with MG132 (20 μM). VP1 was immunoprecipitated with Flag antibody, and ubiquitination of VP1 was detected with HA antibody. (B and C) RNF5 catalyzes the K48-linked VP1 polyubiquitination. HEK293T cells were co-transfected with Myc-RNF5, Flag-VP1 and HA-Ub wild-type (WT) or mutants (K6, K11, K27, K29, K33, K48, K63, K48R, or K63R), and then treated with MG132 (20 μM). VP1 was immunoprecipitated with Flag antibody, and ubiquitination of VP1 was detected with HA antibody. (D) Co-immunoprecipitation analysis of endogenous ubiquitination of VP1. Myc-RNF5 and HA-VP1 were co-transfected into HEK293T cells and then treated with MG132 (20 μM). VP1 was immunoprecipitated with HA antibody, and ubiquitination of VP1 was detected with Ub, K48, and K63 antibodies. (E) RNF5 regulated endogenous ubiquitination of VP1 upon FMDV infection. Lysates of PK-15 cells co-transfected with HA-Ub and Myc-RNF5 or empty plasmids with or without FMDV (MOI = 5) for 8 h in the presence of MG132 (20 μM)…

• 

  Fig 5. Lys200 of VP1 is the ubiquitination site for RNF5. (A) Immunoblot analysis of the ubiquitination of wildtype or mutants VP1 in HEK293T cells. Myc-RNF5, Flag-Ub, and HA-VP1 or mutants were co-transfected into HEK293T cells and then treated with MG132 (20 μM). VP1 or its mutants was immunoprecipitated with HA antibody, and ubiquitination of VP1 or its mutants was detected with Flag antibody. (B) Immunoblot analysis of the degradation of wildtype or mutants VP1 in HEK293T cells. HA-VP1 and three VP1 mutants (K41R, K109R, and K200R) were transfected with or without Myc-RNF5 for 36 h. The degradation of VP1 was visualized by immunoblot. (C) Alignment of VP1 sequences from one representative of each serotype of FMDV. Type A FMDV (GenBank accession no. QZE50273.1); Type Asia I FMDV (GenBank accession no. ACN81751.1); Type C FMDV (GenBank accession no. APX53725.1); Type O FMDV (GenBank accession no. QID89733.1); Type SAT1 FMDV (GenBank accession no. AUN44148.1); Type SAT2 FMDV (GenBank accession no. APX53730.1); Type SAT3 FMDV (GenBank accession no. APX53733.1). (D) Lys200 is on the surface of the virion. The PyMOL visualization created using the FMDV 3D structure from the Protein Data…

• 

  Fig 6. RNF5 deletion enhances the susceptibility of suckling mice to FMDV infection. (A) Knockout schematic representation position of the sequence for RNF5 in RNF5-/- mice. The exon 2–6 and part of intron of RNF5 gene were deleted by CRISPR/Cas9 technology. (B) Expression and protein levels of RNF5 in RNF5-/- and RNF5+/+ mice were detected by PCR amplification and Western blot to determine RNF5 knockout efficiency. (C) Three-day-old RNF5-/- and RNF5+/+ suckling mice were used and injected subcutaneously with 20LD50 FMDV. The survival rates of all group were monitored for 8 days. (D) Viral titers in muscle of RNF5-/- and RNF5+/+ mice challenged with FMDV. (E) FMDV mRNA levels in heart, liver, spleen, lung, kidney, and duodenum of RNF5-/- and RNF5+/+ mice challenged with FMDV (n = 5 per group). Data were obtained from three independent experiments. (F) Images of hematoxylin and eosin staining of heart, liver, spleen, lung, kidney, and duodenum sections from the suckling mice (3 days post-infection, n = 3 per group). Scale bars, 50 μm. Survival rate of mice was examined via log-rank (Mantel-Cox) test (C, n = 6 per group, p = 0.029). Graphs show mean ± SD (n = 3 biological replicates, n =…

• 

  Fig 7. RNF5 pharmacological activator Analog-1 alleviates the pathogenic effects of FMDV in a mouse model. (A) The cell viability was measured and compared with the untreated cell control group. (B-D) PK-15 cells were exposed to varying concentrations of Analog-1 (0, 25, 50, and 100 μM) and subsequently infected with FMDV at 0.1 MOI. Following a 24-hour post-infection period, the levels of viral RNA, viral protein, and viral titers were assessed through RT-PCR (B), immunoblot (C), and TCID50 (D), respectively. (E) C57BL/6 mice were treated with Analog-1 at dosage of 10 mg/kg for seven times at 2-day intervals, then infected with the FMDV at dosage of 30LD50. (F) Survival rates of mice injected with Analog-1 after being challenged with FMDV (n = 7 per group). (G) Representative images of H&E staining of lungs of differently treated mice (3 days post-infection, n = 3 per group). CC50 values were calculated from nonlinear regression plots (n = 2 technical replicates, n = 3 biological replicates in A) from one representative experiment. Graphs show mean ± SD (n = 3 technical replicates, n = 3 biological replicates in B and D) from one representative experiment. Data were analyzed by one-way…

• 

  Fig 8. RNF5 inhibits several picornavirus replication by degrading VP1. (A) VP1 proteins of PV, SVA, and EV71 were degraded by RNF5. HEK293T cells were transfected with VP1 (1 μg) plasmids of PV, SVA, or EV71, along with Myc-RNF5 (1 μg) or Myc-RNF5C42S (1 μg) plasmids. The expression of Myc-RNF5 or Myc-RNF5C42S and Flag-tagged VP1 proteins was detected by immunoblot. (B) RNF5 induces the reduction of several picornavirus VP1 in a dose-dependent manner. HEK293T cells were transfected with VP1 (1 μg) plasmids of PV, SVA and EV71, along with increasing quantities of Myc-RNF5 (0, 1, 2 μg) or Myc-RNF5C42S (2 μg) plasmids. The expression of Flag-VP1 and Myc-RNF5 or Myc-RNF5C42S was detected by immunoblot. (C) Effects of RNF5 knockout on SVA replication. RNF5 knockout 293T cells were infected with SVA (MOI = 1.0) for the indicated times. The cell supernatant and precipitation were collected at 12 or 18 hpi, respectively. Intracellular and extracellular virus mRNA was determined by RT-PCR. (D) RNF5 knockout 293T cells were infected with SVA. At 24 or 36 hpi, the cell mixture, including supernatant and precipitation, was three freeze-thaw cycles, and then the viral titers were determined by…

• 

  Fig 9. A model for RNF5’s anti-FMDV action. This study identifies the E3 ubiquitin-ligase RNF5 as a host factor that interacts with the FMDV VP1 protein to inhibit viral replication. Mutagenesis identified VP1 K200 as the site of ubiquitination by RNF5. A recombinant virus harboring the VP1 K200R mutation replicated to higher titers and exhibited increased pathogenicity in suckling mice. Mice lacking RNF5 exhibit greater histophathological damage to tissues, viral titers and higher mortality following FMDV infection. Treatment of cells with Analog-1 an agonist of RNF5 reduces FMDV replication in cells and i.p. administration to mice protects from FMDV disease. Our results identify RNF5-mediated VP1 protein degradation as a potential therapeutic strategy to treat infections caused by FMDV and related viruses.