POLIOVIRUS INFECTION ENHANCES THE FORMATION OF 2 RIBONUCLEOPROTEIN COMPLEXES AT THE 3' END OF VIRAL NEGATIVE-STRAND RNA

To identify proteins involved in the formation of replication complexe s at the 3' end of poliovirus negative-strand RNA, a combined in vitro biochemical and in vivo genetic approach was used. Five subgenomic cD NA constructs were generated to transcribe different negative-strand R NA fragments. In UV cross-linking assays, distinct differences in bind ing of proteins in extracts from poliovirus-infected and uninfected ce lls to virus-specific, radiolabeled transcripts were observed. Two pro teins present in extracts from poliovirus-infected cells with approxim ate molecular masses of 36 and 38 kDa were shown to cross-link to the 3' end of poliovirus negative-strand RNA. Appearance of the 36- and 38 -kDa proteins in UV cross-linking assays can be detected 3 to 3.5 h af ter infection, and cross-linking reaches maximum levels by 5 h after i nfection. The binding site for the 36-kDa protein overlaps with the co mputer-predicted loop b region of stem-loop I, the so-called cloverlea f structure, and the RNA sequence of this region is required for effic ient binding. Transfection of full-length, positive-sense RNA containi ng a five-nucleotide substitution (positions 20 to 25) in the loop b r egion of stem-loop I into tissue culture cells yielded only viral isol ates with a reversion at position 24 (U-->C). This finding demonstrate s that the wild-type cytidine residue at position 24 is essential for virus replication. RNA binding studies with transcripts corresponding to the 3' end of negative-strand RNA suggest that complex formation wi th the 36-kDa protein plays an essential role during the viral life cy cle.