MARCH8 promotes the proteasomal degradation of foot-and-mouth disease virus VP1, VP2, and VP3 to negatively regulate viral replication

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  Figure 1. MARCH8 overexpression inhibits FMDV propagation. A and B SK6 cells in 6-well plates were transfected with increasing amounts of pCDNA3.1Flag-MARCH8 for 24 h, followed by infection with FMDV O/HN/CHA/93 (MOI = 0.01) for 8 h. VP1 protein levels were assessed by immunoblotting, and virus titres were detected by plaque assay. C and D SK6 cells in 6-well plates were transfected with 2 μg pCDNA3.1Flag or pCDNA3.1Flag-MARCH8 for 24 h and then infected with FMDV O/HN/CHA/93 (MOI = 0.01 or 0.1) for 8 h. VP1 expression was analysed by western blotting, and virus titres were measured by plaque assay. E and F SK6 cells in 6-well plates were transfected with 2 μg pCDNA3.1Flag or pCDNA3.1Flag-MARCH8 for 24 h and then infected with 0.01 MOI FMDV O/HN/CHA/93 for the indicated times. Viral replication was determined by western blotting and plaque assays. G SK6 cells in 6-well plates were transduced with shNC or shMARCH8 lentivirus for 48 h, and RNA was extracted from the cells for RT‒qPCR analysis. H‒J SK6 cells in 6-well plates were transduced with shNC or shMARCH8 lentivirus for 48 h and then infected with FMDV (MOI = 0.01 or 0.1) for 8 h. VP1 protein levels were analysed by western blotting,…

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  Figure 2. MARCH8 interacts with VP1, VP2 and VP3. A HEK293T cells were co-transfected with 1 μg PCAGGS-HA, HA-VP1, HA-VP2, HA-VP3, HA-3C, or HA-3D and 1 μg pCDNA3.1Flag-MARCH8 for 24 h. The resulting cellular lysates were incubated for co-IP analysis. B HEK293T cells were co-transfected with 1 μg of PEBG-GST, GST-VP4, GST-3A, GST-3B, or GST-2B and 1 μg of pCDNA3.1Flag-MARCH8 for 24 h. The resulting cellular lysates were incubated for co-IP analysis. C HEK293T cells were co-transfected with 1 μg of PEGFP-C1 or GFP-2C and 1 μg of pCDNA3.1Flag-MARCH8 for 24 h. The resulting cellular lysates were incubated for co-IP analysis. D HEK293T cells were co-transfected with 1 μg pCDNA3.1Flag or pCDNA3.1Flag-MARCH8 and 1 μg HA-VP1, HA-VP2, or HA-VP3 for 24 h. The resulting cellar lysates were incubated for co-IP analysis. E Confocal assays of HEK293T cells transfected with 0.4 μg PCAGGS-HA, HA-VP1, HA-VP2, or HA-VP3 and 0.2 μg pCDNA3.1Flag-MARCH8 for 24 h, followed by fixation and staining with anti-Flag (green), anti-HA (red) and DAPI nuclear stain (blue). Images were captured by confocal microscopy. F and G SK6 cells were transfected with 0.5 μg pCDNA3.1Flag or pCDNA3.1Flag-MARCH8 for 24 h and then…

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  Figure 3. MARCH8 degrades VP1, VP2, and VP3 via the proteasome pathway. A, D and G HEK293T cells were co-transfected with 0.4 μg of HA-VP1, HA-VP2, or HA-VP3 and increasing amounts of pCDNA3.1Flag-MARCH8 for 24 h. The resulting cellular lysates were analysed by immunoblotting. B, E and H SK6 cells were co-transfected with 0.8 μg of HA-VP1, HA-VP2, or HA-VP3 and increasing amounts of pCDNA3.1Flag-MARCH8 for 24 h. The resulting cellular lysates were analysed by immunoblotting. C, F and I HEK293T cells were co-transfected with 0.4 μg pCDNA3.1Flag or Flag-MARCH8 and 0.4 μg HA-VP1, HA-VP2, or HA-VP3 for 12 h, followed by treatment with DMSO, NH4CL (10 mM), ZVAD (50 μM), or MG132 (10 μM) for 12 h. The resulting cellular lysates were analysed by immunoblotting.

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  Figure 4. MARCH8 induces the ubiquitination of VP1, VP2 and VP3. A HEK293T cells were co-transfected with 1 μg pCDNA3.1Flag or pCDNA3.1Flag-MARCH8, 1 μg pCDNA3.0-HA-UBI or 1 μg PEBG-GST, GST-VP1, GST-VP2, or GST-VP3 for 24 h. The resulting cellular lysates were analysed by co-IP. B Schematic representation of ubiquitin (UBI) and nine UBI mutants (K6, K11, K27, K29, K33, K48, K63, KR and K33R). C–E HEK293T cells were co-transfected with 1 μg of pCDNA3.1Flag-MARCH8, 1 μg of pCDNA3.0-HA-UBI (wild type or mutants), and 1 μg of PEBG-GST, GST-VP1, GST-VP2, or GST-VP3 for 24 h. The resulting cellular lysates were analysed by co-IP. F HEK293T cells were co-transfected with 1 μg of pCDNA3.1Flag-MARCH8, 1 μg of pCDNA3.0-HA-UBI mutants (K33 and K33R), or 1 μg of PEBG-GST, GST-VP1, GST-VP2, or GST-VP3 for 24 h. The resulting cellular lysates were analysed by co-IP.

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  Figure 5. MARCH8 mediates the degradation of VP1, VP2, and VP3 at specific sites. A, C and E Sequence alignment of VP1, VP2, and VP3 from different FMDV serotypes (type-A: DQ533483.2; type-C: AM409325.1; type-Asia: MZ493234.1; type-SAT 1: MN275121.1; type-SAT 2: MZ090097.1; type-SAT 3:MG372727.1). B, D and F HEK293T cells were co-transfected with 0.4 μg pCDNA3.1Flag-MARCH8 and 0.4 μg wild-type or mutant VP1, VP2, or VP3 for 24 h. The resulting cellular lysates were analysed by immunoblotting. G HEK293T cells were co-transfected with 1 μg pCDNA3.1Flag-MARCH8 and 1 μg wild-type or mutant VP1, VP2, or VP3. After 24 h, the cellular lysates were analysed by co-IP. H HEK293T cells were co-transfected with 1 μg pCDNA3.1Flag-MARCH8, 1 μg pCDNA3.0-HA-UBI (wild type or mutant), and 1 μg GST-VP1K210R, GST-VP2K63R, or GST-VP3K118R for 24 h. The cellular lysates were analysed by co-IP.

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  Figure 6. MARCH8-induced degradation of VP1, VP2, and VP3 is dependent on its W112 site. A Schematic diagram of MARCH8 and its mutant. B–D HEK293T cells were co-transfected with 0.4 μg pCDNA3.1Flag-MARCH8 or pCDNA3.1Flag-MARCH8W112A and 0.4 μg HA-VP1, HA-VP2, or HA-VP3 for 24 h. The cellular lysates were analysed by immunoblotting. E–G HEK293T cells were co-transfected with 1.0 μg pCDNA3.1Flag-MARCH8 or pCDNA3.1Flag-MARCH8W112A and 1.0 μg HA-VP1, HA-VP2, or HA-VP3 for 24 h. The resulting cellular lysates were analysed by co-IP.

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  Figure 7. MARCH8 inhibits FMDV replication by its ZF and TM domains. A and B SK6 cells in 6-well plates were transfected with 2 μg pCDNA3.1Flag-MARCH8 or pCDNA3.1Flag-MARCH8W112A for 24 h, followed by infection with 0.01 MOI FMDV O/HN/CHA/93 for 8 h. Viral replication was determined by western blotting and plaque assays. C Schematic representation of MARCH8 and its mutant. D and E SK6 cells in 6-well plates were transfected with 2 μg pCDNA3.1Flag-MARCH8 or pCDNA3.1Flag-△ZF + TM for 24 h, followed by infection with 0.01 MOI FMDV O/HN/CHA/93 for 8 h. Viral replication was determined by western blotting and plaque assays. The data are shown as the means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  Figure 8. Schematic model showing how MARCH8 negatively regulates FMDV infection. MARCH8 inhibits FMDV replication by degrading the viral structural proteins VP1, VP2, and VP3 in a proteasome-dependent manner. The E3 ubiquitin ligase activity of MARCH8 is required for the degradation of VP1, VP2 and VP3. MARCH8 mediates K33-linked polyubiquitylation of VP1, VP2 and VP3. In addition, the VP1K210R, VP2K63R, and VP3K118R substitutions cannot be degraded by MARCH8.