We have followed the viral processing cascade and polyprotein precursor fates during encephalomyocarditis virus (EMCV) infection of HeLa cells using a panel of monoclonal antibodies (mAbs). Within the first 2-4 h of infection, signals of antibodies specific for the 2A, 3B(VPg), 3C(pro) and 3D(pol) proteins were found to co-localize in nucleoli at the rRNA synthesis and cellular protein B23 (nucleophosmin) sites. Cellular fractionation identified viral protein precursor 3BCD as the common source of the P3-region antibody signals. Previously thought to be a minor product of the polymerase region cleavage pathways, the nuclear targeting of this precursor was localized with engineered mutations to five P2 and P3 region polyprotein processing sites. A nuclear localization motif (NLS), similar to that in many yeast ribosomal proteins, was identified near the N-terminus of the 3D(pol) sequence. Point mutations within this motif prevented nuclear and nucleolar localization by all forms of 3B(VPg), 3C(pro) and 3D(pol), and were lethal to the virus because they also prevented genome replication. However, viral RNA synthesis was not required for nucleolar transport and 3BCD was found in nuclei, even when the 3D(pol) was inactivated. Co-immunoprecipitation experiments showed a tight association between 3BCD and B23 (nucleophosmin), suggesting a possible ribosomal protein-like mechanism for nuclear transport. Infected cell extracts analyzed with microarrays, quantitative slot-blots and pulse-labeling experiments confirmed a nearly complete shutoff of host pol-II-dependent mRNA synthesis during EMCV infection, in reactions that depended on wild-type 2A protein. In contrast to human rhinovirus-16 infection, rRNA synthesis by pol-I and pol-III were not turned off by EMCV, although the cellular concentration of rRNA decreased during infection, relative to control samples. The data suggest that nuclear targeting by 2A and 3BCD may be responsible for regulating cellular mRNA and rRNA transcription during infection, perhaps via a proteolytic mechanism catalyzed by the endogenous 3C(pro) sequence.