Studies on Dicistronic Polioviruses Implicate Viral Proteinase 2Apro in RNA Replication

A dicistronic poliovirus W1-P1/E/P2,3-1 with the genotype [PV]5'NTR-P1-[EMCV]IRES-[PV]P2,3-3'NTR (Molla, Jang, Paul, Reuer, and Wimmer, 1992, Nature 356, 255) was used to investigate whether the viral proteinase 2Apro, whose primary function in proteolytic processing was erased through the insertion of an internal ribosomal entry site (IRES) element into the ORF of the polyprotein, had other function(s) in viral replication. Deletion of 2Apro from W1-P1/E/P2,3-1 rendered the corresponding transcripts unable to replicate whereas partial deletion of 2Apro or an exchange of Cys109 (an amino acid of the catalytic triad of the proteinase) to Ala reduced RNA replication. No cytopathic effects were observed after transfection with any of the three dicistronic constructs containing mutant 2A, and no virus was recovered after attempts to expand a possibly low yield of mutant virus. In contrast, insertion of the IRES of encephalomyocarditis virus (EMCV) into the ORF of the poliovirus polyprotein at the cleavage site between 2Apro and 2B yielded the novel dicistronic virus W1-P1,2A/E/2BC,P3-1 with the genotype [PV]5'NTR-P1-2A-[EMCV]IRES-[PV]2BC-P3-3'NTR, expressing a small plaque phenotype. These results indicate that neither the intact P2 polypeptide nor the cleavage fragment 2AB of P2 is required for viral proliferation. On the other hand, 2Apro appears to be an essential component in RNA replication as no viral RNA synthesis can be observed by reverse transcription/PCR in cells transfected with dicistronic RNA lacking this viral polypeptide.