EXPRESSION OF POLIOVIRUS P3-PROTEINS USING A RECOMBINANT VACCINIA VIRUS RESULTS IN PROTEOLYTICALLY ACTIVE 3CD PRECURSOR PROTEIN WITHOUT FURTHER PROCESSING TO 3C(PRO) AND 3D(POL)

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is sub sequently processed by viral encoded proteases. The initial proteolyti c cleavages result in the production of a Pl polyprotein which contain s the capsid proteins, and the P2 and P3 polyproteins which contain pr oteins required for replication. The P3 polyprotein consists of the 3A B protein (containing the viral genome-linked protein, VPg), the viral protease, 3C(pro), and RNA polymerase, 3D(pol). To further study the expression and proteolytic processing of poliovirus P3 proteins in viv o, we have utilized recombinant vaccinia virus vectors to express nucl eotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus W-P3 exhibit ed in vivo proteolytic activity as evident by processing of the polypr otein to generate the 3CD protein, consisting of a fusion between the 3C(pro) and 3D(pol) proteins. Further processing of the 3CD protein to 3C(pro) and 3D(pol), however, was not detected in cells infected with W-P3. Subcellular fractionation of W-P3-infected cells demonstrated t hat the 3CD protein was present in both the soluble and membrane fract ions. Finally, the 3CD protein expressed from W-P3 was stable in cells co-infected with W-P3 and poliovirus and no further processing to 3D( pol) was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3D(pol) in poliovirus-infected cells.