Abstract
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.
Highlights
Infection of cells with poliovirus results in a rapid poliovirus infection is thought to result from specific inactiinhibition of host RNA and protein synthesis.Concor- vation of transcription factors required for RNA polymerase dant with this shutoff, the p220 subunit of the capbindingprotein complex is cleaved, probablyindirectly, by the poliovirusproteinase p2A (2Apr0).To elucidate themechanism of action of 2APr0in inhibiting protein synthesisin vivo, we studied the effect of tran
The specific inhibition of protein synthesis upon poliovirus infection is thought to result from proteolytic inactivation of the large subunit (p220) of the cap-binding protein complex, sientexpression of 2APr0in COS-1 monkey kidney eIF-4F,' mediated by the poliovirus protease 2APr0(9, 10)
Expression of ZAP" on COS-1Cells-The 2AP" coding region from the poliovirus type I Sabin strainwas modified by siteanalyzed for protein synthesis by ["Slmethionine pulse labeling and preparation of cell extracts as described under "Experimental Procedures." Lanes 4-7 represent total protein synthesis analyzed by directed mutagenesis to introduce initiation and termination SDS-PAGE
Summary
RNA blots were hybridized to probes prepared by polypeptide, a GGC codon at base pair 3829 of the poliovirus genome nick translation of the DHFR, p-actin [31], or ZAP'" coding regions. Samples of 5-50 pg of total phatase Another 10-pg aliquot of p2A was digested with EcoRI and RNA were annealed to 25-50 fmol of 32P-end-labeledoligonucleotide treated with calf intestine phosphatase. Both DNA preparations were (2 X lo' cpm/pmol) at 30 "C overnight. The filter was washed at room temperature for 10 min in each filtered onto nitrocellulose dots using a dotblot apparatus (Schleicher of the following: once with TBS, three times with TBS containing and Schuell).
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