PhD

dissertationPoliovirus RNA synthesis in vitro was studied in an attempt to determine the conditions required for accurate product synthesis. Analysis of in vitro reaction products by gel electrophoresis, sucrose gradient sedimentation, hybridization and RNase T(,1) oligonucleotide fingerprints revealed that crude cytoplasmic extracts synthesize products which are identical to those made in infected cells; whereas, detergent-extracted complexes fail to synthesize full-length single-stranded RNA. Both complexes synthesize predominantly plus-stranded RNA, and detergent treatment inhibits synthesis of full-length single-stranded RNA by complexes which, untreated, are capable of making this product. The results suggest that poliovirus replication complexes consist of a detergent-resistant core complex which elongates nascent chains on predominantly double-stranded templates, and a detergent-sensitive factor which permits completion and release of full-length single-stranded RNA. RNase T(,1) oligonucleotide fingerprints of in vitro products revealed that detergent-extracted complexes fail to replicate a specific internal region of the RNA, suggesting that detergent interferes with a function involved in conformational aspects of replication. A short-lived replication complex protein, 4a, was identified which is associated with the detergent-extracted complex. Tryptic peptide analysis, pulse-chase data and the effects of inhibitors of proteolytic enzymes suggest that 4a may be a precursor to the protein associated with RNA elongation activity, 4b. Since VPg may be a primer for RNA synthesis and appears to be derived from a precursor protein, 4a may contain the sequences for both VPg and 4b. Thus, 4a may be involved in initiation of RNA synthesis. This possibility prompted an analysis of initiation by replication complexes in vitro. Crude cytoplasmic extracts were demonstrated to be capable of utilizing an exogenous RNA template to synthesize minus-strand RNA, which was detected among double-stranded products by RNase T(,1) fingerprints. For comparative purposes, a fingerprint of poliovirus minus-strand RNA from infected cells was prepared. This approach may be useful in the development of an in vitro assay which will permit further analysis of possible initiation activity by the replication complex-associated protein, 4a