Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap-binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap-independent translation of 2Apro. Expression of this mRNA was also reduced 25-fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation.