Poliovirus RNA Replication and Genetic Complementation in Cell-Free Reactions

Abstract

Poliovirus RNA replicates in membrane-associated replication complexes in the cytoplasm of infected cells. By using a reversible inhibitor of poliovirus RNA replication, it is possible to synchronize viral RNA replication. The processing of the viral polyprotein results in the formation of the individual viral proteins along with stable intermediates in the processing pathway. To expand the utility of the in vitro complementation assay, experiments were designed to determine if all of the viral replication proteins could be provided in trans to support the replication of mutant RNA templates. The authors engineered two transcript RNAs (DJB2 and DJB15) that contained large out-of-frame deletions in the polyprotein coding sequence. The results to date using the in vitro complementation assay indicate that the 5' cloverleaf, the 3' nontranslated region (NTR), and the poly(A) tail are the minimum sequences required for negative-strand synthesis. Previous studies have shown that the 5' cloverleaf plays an important role in viral RNA replication. To investigate the role of the 5' cloverleaf in negative-strand synthesis, the authors determined how cloverleaf mutations affected negative-strand synthesis in preinitiation RNA replication complexes. Results of these experiments showed that 5' cloverleaf mutations dramatically diminished RNA stability and negative-strand RNA synthesis. The results of recent studies indicate that the 5' cloverleaf is required for the initiation of negative-strand synthesis. Once the viral mRNA is cleared of translating ribosomes, it could then serve as a template for negative-strand synthesis.