A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence

Figures

• 

  Figure 1. Effect of cysteine 38 of protein 3A at the ultrastructural level. (A) HeLa cells were infected with GFP-CVB3 encoding protein 3A wt or 3A[C38A] at MOI 1 for 16 h and processed for the immunoblotting of protein 3A. (B) Fractionation of non-infected cells and cells infected with GFP-CVB3 variants (3A wt and 3A C38A) at MOI 1 for 5 h. Pre-cleared lysates in hypotonic lysis buffer were subjected to differential centrifugation at 16,000 rcf (16 k) and 120,000 rcf (120 k). Resulting membrane pellets and supernatants were analyzed by the immunoblotting of 3A-interactor ACBD3, loading control GAPDH. Quantification of indicated immunosignals in 16k-rcf membrane pellets (n = 4). n.s.—not significant. (C–G) Thin-section electron microscopy of HeLa-cells after infection with either GFP-CVB3-3A wt or GFP-CVB3-3A[C38A]. (C,D) Non-infected cells (control). (E–H) Cells infected with GFP-CVB3-3A wt (E,F) or GFP-CVB3-3A[C38A] (G,H). Images in the right column show a detail of the overview images in the left column. Both viruses induce the formation of large virus factories, which are composed of membrane-enveloped replication organelles (ro) and dense cytoplasmic deposits (arrows) that are most…

• 

  Figure 2. Cysteine 38 of protein 3A affects the subcellular distribution of the viral 3A protein. (A) Confocal microscopy of HeLa cells infected with GFP-CVB3 variants at MOI 1 for 5 h. GFP signal reflecting CVB3 replication, staining of DNA (DAPI, blue), and protein 3A (magenta). Scale bar: 10 µm. (B) Quantitative comparison of anti-3A immunofluorescence intensity distributions (n = 15) depicted in (A). (C) Autocorrelation-based image correlation spectroscopy of anti-3A immunofluorescence shown in (A). Two-dimensional plots depicting the auto-correlation of x/y-shifted confocal images to assess cluster sizes, which are collapsed into a 1D Pearson correlation coefficient plot (n = 15). In order to quantitatively compare the immunofluorescence frequency distributions and auto-correlation curve of the two strains, differences between single bins from GFP-CVB3 3Awt and GFP-CVB3 3A[C38A] were tested with a one-sample t-test against zero, * p < 0.05. We used a significance level of 0.05 and Bonferroni correction to correct for the multiple testing of several bins.

• 

  Figure 3. Cysteine 38 of protein 3A supports virus replication. (A) HeLa cells were infected with GFP-CVB3 encoding wt protein 3A, 3A[C38A], or 3A[C38S] at MOI 1 for 5 h. Virus titers were determined by plaque assay. (B) HeLa cells were infected with GFP-CVB3 variants at MOI 1 for 5 h. Fluorescence microscopy with the 488 nm excitation of infected cells. (C) Analysis by flow cytometry to assess the percentage of GFP positive cells and mean fluorescence intensity (MFI) of GFP in GFP-positive cells. (D) Disulfide-linked enteroviral protein 3A promotes viral replication. HeLa cells were infected with GFP-CVB3 encoding protein 3A wt or 3A[C38A] at MOI 0.01 for the indicated periods. Fluorescence microscopy at 30× magnification with 488 nm excitation of infected cells. (E) The amount of infectious virus was determined by plaque assay (n = 3). The bar charts depict the actual viral titers and normalized titers, respectively. (F) Fractionation of non-infected cells and cells infected with GFP-CVB3 variants at MOI 1 for 5 h. Pre-cleared lysates in hypotonic lysis buffer were subjected to differential centrifugation at 16,000 rcf (16 k) and 120,000 rcf (120 k). Resulting membrane pellets and…

• 

  Figure 4. Mitigation of virus-triggered liver pathology by the CVB3-3A[C38A] mutant. Male C57BL/6 mice were infected intraperitoneally with 1 × 105 pfu of CVB3 (wt) encoding wt protein 3A or 3A[C38A] and sacrificed at day 3 after infection (n = 10 for each virus strain). (A) Virus titers in indicated tissues were determined by a plaque assay. (B) Normalized body weight change (day 0 = 100%) at each day after infection. (C) Body temperature at baseline and 3 days after infection. (D) Serum glucose levels at baseline (control) and 3 days after infection. (E) Periodic acid-Schiff (PAS) staining of liver sections. (F) Graph depicts scoring of staining intensity (0–4, 0 = PAS-negative cells, 4 = PAS-positive cells). (G) Serum alkaline phosphatase levels 3 days after infection (n = 10 per group). (H) Hematoxylin-eosin staining of liver sections. (I) Liver pathology was categorized based on the severity of inflammation and necrosis using the following scoring system: 0: no inflammation/ necrosis; 1: scattered immune cells/mild necrosis (<10%); 2: immune cell foci/marked necrosis (10–50%); 3: diffuse immune cell infiltrates/severe necrosis (>50%), according to [23]. Unpaired t-test with the…