Egress-enhancing mutation reveals inefficiency of non-enveloped virus cell exit

Figures

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  Figure 1.. Selection to enrich for fast-replicating MNV mutants. (A) Schematic showing serial passage strategy. Initial MNV3 stock (P0) was used to infect BV2 cells, and progeny were harvested at 6 hours or 24 hours post-infection. Low titer viral progeny harvested at 6 hours were amplified overnight in BV2 cells to facilitate subsequent passages. Half of the volume of each sample was used to initiate a new passage, and the cycle was repeated 11 times. (B) Viral infection assay comparing the initial stock virus (Passage 0) and viruses passaged 11 times, collecting at 6 hours vs. 24 hours. BV2 cells were infected at an MOI of 0.1 for 6 hours. Cells and supernatant were pooled for each sample, and viral titers were quantified via plaque assay. Data are mean ± SEM (n=6, ≥ 3 independent experiments). *P<0.05, one-way ANOVA. (C) Single-cycle growth curve of Passage 0 and Passage 11–6 hrs. BV2 cells were infected at MOI 0.1 and cells and supernatant were collected and viral titer determined via plaque assay. Data are mean ± SEM (n=7, ≥ 3 independent experiments). *P<0.05, unpaired t test. LOD, limit of detection.

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  Figure 2.. NS3-K40R is sufficient for the faster MNV3 replication phenotype. (A) Schematic of MNV3 genome with mutations found after eleven passages. Consensus sequence analysis revealed three mutations in viral progeny from passage 11–6 hr stocks: Two amino acid substitutions in NS3, K40R and I340T, and a silent mutation in the minor capsid protein VP2. Passage 11–24 hr stocks also contained the NS3-I340T mutation. (B) Viral infection assay of MNV3 WT, NS3-K40R and VP2–114. Infectious clone-derived viruses bearing NS3-K40R, the VP2–114 silent mutation, or no engineered mutations (WT) were used to infect BV2 cells at an MOI 0.1. Cells and supernatant were collected, and virus was quantified via plaque assay. Data are presented as mean ± SEM (n ≤ 4; ≥ 4 independent experiments). *P<0.05, two-way ANOVA. (C) Emergence of adenine to guanine (AAG to AGG) mutation at position 1147 (NS3 amino acid 40) during viral passaging. The substitution of adenine with guanine changed coding from lysine to arginine. Peak heights were determined from chromatograms of the consensus sequence across passages. The percentages of peak heights were calculated based on the relative abundance of all four nucleotides…

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  Figure 3.. NS3 K40R does not alter RNA synthesis or NTPase activity. (A) Quantification of viral RNA during infection. BV2 cells were infected with WT or NS3-K40R at MOI 2, and viral RNA at indicated time points was harvested and quantified via RT-qPCR targeting MNV ORF1. Data are mean ± SEM (n=3, ≥ 3 independent experiments). *P<0.05, two-way ANOVA. (B) Purified MBP-NS3 proteins used for NTPase assay. MBP-NS3 fusion proteins or MBP protein alone were purified and analyzed by 4–20% SDS-PAGE, followed by Coomassie Blue staining. The bands of proteins were quantified using ImageJ. (C) NTPase activity of purified NS3 proteins. NS3-MBP fusion proteins or MBP protein alone were incubated with 10 μM ATP for 30 min at 37°C, and inorganic phosphate released from ATP hydrolysis was quantified via a colorimetric assay. Data are mean ± SEM (Representative data from three independent protein preparations). *P<0.05, ANOVA.

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  Figure 4.. NS3-K40R increases extracellular viral titers but not cell-associated viral titers. BV2 cells were infected with MNV3 WT or NS3-K40R at MOI 0.1 for 4, 6 and 8 hrs post-infection, and progeny were harvested from (A) cells (representing virion particles within cells) or (B) culture supernatants (representing extracellular virus) followed by plaque assay quantification. Data are mean ± SEM (n=3, ≥ 3 independent experiments). *P<0.05, two-way ANOVA. LOD, limit of detection.

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  Figure 5.. NS3-K40R induces increased cell death, mitochondrial dysfunction, and membrane disruption. (A) NS3-K40R increases cell death at early times post-infection. BV2 cells were infected with WT or NS3-K40R at MOI 2 and at 6 or 8 hours post-infection the number of dead cells was quantified by flow cytometry to detect Sytox nucleic acid staining of non-viable cells. Values represent the subtraction of the percentage of mock-treated cells. Data are mean ± SEM (n=3, ≥ 3 independent experiments). *P<0.05, two-way ANOVA. (B) NS3-K40R increases mitochondrial superoxide at early times post-infection. BV2 cells were infected with WT or NS3-K40R at MOI 2 and at 6 or 8 hours post-infection mitochondrial superoxide was quantified by flow cytometry to detect MitoSOX staining. Values represent the subtraction of the percentage of mock-treated cells. Data are mean ± SEM (n=3, ≥ 3 independent experiments). *P<0.05, two-way ANOVA. (C-D) Cardiolipin liposome leakage assays with purified-tagless NS3 proteins. (C) Liposome leakage assay with low concentrations of NS3 proteins reveals increased activity of NS3-K40R compared with NS3-WT. Cardiolipin liposomes (0.1 mM; 80% phosphatidylcholine, 20%…

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  Figure 6.. NS3-K40R increases viral titers in STAT1 −/− mice. STAT1−/− mice were perorally infected with 106 PFU of WT and NS3-K40R followed by assessment of (A) weight loss, (B) survival until 8 days post-infection. Data are mean ± SEM (n=8, ≥ 2 independent experiments). *P<0.05, Mantel-Cox test. Infections were repeated, but mice were sacrificed at 24 hours post-infection for viral titer analysis via plaque assay for tissues including (C) spleen, (D) duodenum, (E) ileum, and (F) colon. Data are mean ± SEM (n=12, ≥2 independent experiments). *P<0.05, unpaired t test. LOD, limit of detection. LOD was determined based on the average tissue weight.