Effect of conservative mutations of residues Asp-431 and Asp-391 on
pVP2/VP2 proteolytic maturation. A, Western blot analysis of
proteins expressed in cells infected with rVV VT7LacOI/POLY, VT7LacOI/D431N,
or VT7LacOI/D391N, respectively. Infected cultures were harvested at 24, 48,
and 72 h pi, respectively. The corresponding extracts were subjected to
SDS-PAGE and Western blot using anti-VP2 serum. B, QM7 cells infected
with rVV VT7LacOI/POLY, VT7LacOI/D431N, or VT7LacOI/D391N were metabolically
pulse-labeled with [35S]Met for 1 h at 19 hpi (hpi). The
radioactivity was chased with by adding fresh medium containing an excess of
cold methionine. Cultures were harvested at 0, 24, 48, and 72 h post-labeling,
and the corresponding extracts were immunoprecipitated using anti-VP2 serum.
The resulting samples were subjected to SDS-PAGE followed by autoradiography.
Molecular weight markers (expressed in kDa, left) and bands
corresponding to proteins pVP2 and VP2 (right) are indicated.