The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2at, as revealed by their reaction with specific antibody.Poliovirus, a member of the Picornavirus family, contains a single-stranded RNA genome of positive polarity. This 7.5-kilobase RNA includes 5' and 3' untranslated regions (UTRs) of 745 and 73 nucleotides, respectively, whose functions are beginning to be revealed (reviewed in ref. 1). Analysis of the 5' UTR by site-directed mutagenesis indicates that it plays an important role in RNA replication and translation. For example, recently it was shown that ribosomes bind internally to sequences within the 5' UTR (2). Viral mutants with alterations in this region have been isolated that are defective in virus RNA replication (3,4). This complex region also contains major determinants that attenuate neurovirulence (5-7). These functions are likely to be mediated by the interaction of cellular polypeptides with the 5' UTR.To determine whether cell and/or viral polypeptides interact with the poliovirus 5' UTR, a mobility-shift assay was used to examine cytoplasmic extracts for their ability to form complexes with defined regions of the RNA. These studies reveal specific binding of cell factors with two sequences within the 5' UTR. One of these regions of binding was more precisely mapped by footprinting with a chemical nuclease.The translation initiation factor eIF-2a (eukaryotic initiation factor 2a) participates in complex formation with either sequence. However, competition analysis and determination of salt requirements for assembly of the complexes suggest that unique cellular factors are present in each complex. These RNA-protein interactions probably represent the initial steps in poliovirus replication. MATERIALS AND METHODSCells, Viruses, and Recombinant DNAs. HeLa S3 cells were maintained as monolayers in Dulbecco's minimal essential medium containing 10% (vol/vol) horse serum as described (8). For preparation of infected cell extracts, HeLa cells were infected at a multiplicity of infection of 100 with the P1/ Mahoney strain of poliovirus. Poliovirus type 2 (P2/Lansing) cDNAs used for in vitro RNA synthesis (Fig. 1) have been described (9).Preparation of Extracts. Confluent monolayers of mockinfected or poliovirus-infected cells 1-3 hr postinfection were scraped into 100 mM NaCI/1 mM EDTA...

Poliovirus-infected cells contain a previously unrecognized particle which appears to be an intermediate in virion synthesis and therefore has been named proviron. It sediments at about 125S, contains the three procapsid proteins, VP-O, VP-1, and VP-3, and has 35S viral RNA. It is disrupted both by sodium dodecyl sulfate and EDTA but the RNA resists digestion by ribonuclease. Pulsechase experiments and studies employing the virus-specific inhibitor, guanidine, all indicate that the proviron is formed by combination of newly made RNA with the procapsid. Cleavage of VP-0 to form VP-2 and VP-4 follows formation of the provirion and would be the final step in poliovirus morphogenesis.

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Formation of an apparent virion precursor, the provirion, can be demonstrated in cytoplasmic extracts of poliovirus-infected HeLa cells.