Summary of the design and cell culture characterization of NS7 mutants

Mutant in orthologous viral polymeraseaFidelity in orthologous virus polymerasebMNV NS7 mutantcMNV straindViable in MNVeReplication in cell culturefReplication in vivog
PV G64SHighV65S(1)No
P72S(1, 3)YesModest increaseModest increase
E75S(1, 3)YesModest increaseWT
FMDV P169S/CV S164PHigh/lowK174R(3)YesWTWT
K174S(3)YesSignificantly decreasedSignificantly decreased
CV S298TLowS313T(3)YesModest decreaseSignificantly decreased
CV A372VLowI391D(3)No
I391L(3)YesWTSignificantly decreased
Isolated by passage in ribavirinT35I(3)YesWTModest decrease (viral RNA)
Isolated by passage in ribavirinV330I(3)YesWTWT
Designed based on MNV V330IV330A(3)No
  • a Substitutions generated in this study were based on fidelity changes found in other viruses (4, 5, 29, 31, 32): G64S in PV; P169S in FMDV; and S164P, S298T, and A372V in CV.

  • b Effect on virus polymerase fidelity caused by substitutions shown in the first column.

  • c MNV NS7 polymerase mutants generated based on the fidelity of the residues shown in the first column. Mutants V330I and T35I were constructed after isolation of these mutations in MNV populations treated with increasing concentrations of ribavirin (see Fig. S1 in the supplemental material). Other variants of position 330 (V330A or -S) were also prepared based on the isolation of V330I during ribavirin treatment. These mutants were not viable.

  • d MNV strain. NS7 mutations were introduced in plasmids containing full MNV-1 or MNV-3 genome sequences. Hence, “(1)” denotes MNV-1, “(3)” denotes MNV-3, and “(1, 3)” denotes those mutations tested in both strains. For the experiments represented in the figures, only MNV-3 variants were used.

  • e The viability of NS7 mutants was assessed by reverse genetics after recovery in BHK-21 (or BHK-21-derived BSR-T7) cells followed by 3 serial passages in RAW264.7 cells as described in reference 27.

  • f Phenotype in cell culture. “Modest increase or decrease” (in virus replication) indicates statistically significant changes in virus titers of ≤1 log10 (two-way analysis of variance [ANOVA] test) at any given time point during replication kinetics. “Significantly decreased” in virus replication (K174S) refers to statistically significant changes in virus titers of ~2 log10 at an early replication time point. In this column, “P72S” and “E75S” refer to the corresponding NS7 mutants in the MNV-3 genome context.

  • g Phenotype in vivo. “Modest increase” (in virus replication) in vivo indicates a highly significant change in the titer of virus shed in feces (>1 log10; P < 0.001; two-way ANOVA). “Significantly decreased” indicates highly significant changes in levels of both viral RNA and infectious virus shed in feces (P < 0.001; ANOVA test). “Modest decrease (viral RNA)” indicates significantly lower RNA levels but not virus titers shed in mouse feces. In this column, “P72S” and “E75S” refer to the corresponding NS7 mutants in the MNV-3 genome context.