Table 2.
Effect of mutations in the MNV VPg on infectivity, activities, and stability
| Mutation | Virus recoverya | NS7pol enhancementb | VPg nucleotidylationc | VPg core stabilityd |
|---|---|---|---|---|
| Wild type | +++ | + | + | + |
| N-terminal tail | ||||
| K3A | − | + | + | ND |
| K5A | ++ | + | + | ND |
| K7A | ++ | + | + | ND |
| L21A | − | + | + | ND |
| α-helix 1 | ||||
| D23A | − | − | − | ND |
| E25A | − | − | − | ND |
| Y26A/Fe | − | − | − | + |
| D27A | + | − | − | ND |
| E28A | − | − | − | ND |
| F29A | − | − | − | − |
| R32D | ++ | + | + | +/− |
| Loop | ||||
| R36D | ++ | + | + | ND |
| Y40A | − | − | − | − |
| Y40F | +++ | + | + | +/− |
| S41A | +++ | + | + | ND |
| α-helix 2 | ||||
| D43A | +++ | + | + | ND |
| Y45A | − | +/− | + | − |
| Y45F | − | − | − | + |
| D48R | +++ | − | − | +/− |
| R32D/D48R | ++ | ND | ND | +/− |
| C-terminal tail | ||||
| V115A | ++ | + | + | ND |
| D116A | ++ | + | + | ND |
| Y117F | +++ | + | + | ND |
Recovery is expressed as the yield posttransfection relative to that of the wild type (+++) as assayed by >3 independent experiments. Typical yields of wild-type virus were 1 × 104 to 5 × 104 TCID50 units. −, no virus detected; +, up to 100 TCID50 detected; ++, up to 1,000 TCID50 detected; +++, up to WT levels of virus detected.
Enhancement of RNA synthesis by NS7pol in the luciferase reporter assay is denoted as a plus (+) when the luciferase reporters are statistically above the signal from NS7pol expressed without VPg at a P value of >0.05 in the Student t test.
VPg nucleotidylation is derived from the Western blots in Fig. 7.
VPg core stability was assessed from 1H NMR shift data in Fig. 6A. The WT VPg core contains nine distinct peaks, and samples with 8 or more peaks are denoted with a +. Samples with three to five peaks are denoted with a +/−, and samples with less than three peaks are denoted with a −. ND, not determined.
Infectivity data are for Y26F; other assay data are for Y26A.