Prof. James B. Flanegan

Positive-strand RNA viruses include a large number of important pathogens that cause a wide variety of diseases. Diseases associated with these viruses include poliomyelitis, hepatitis, the common cold, aseptic meningitis, encephalitis, and myocarditis. Poliovirus (PV) is a member of this group of viruses and serves as the prototypic virus for most genetic and biochemical studies on positive-strand RNA viruses. The primary objective of my research is to characterize the molecular mechanisms that are involved in the replication of picornavirus RNA and other positive-strand RNA viruses. We have developed a cell-free RNA replication system to investigate picornavirus RNA replication. The synthesis of authentic VPg-linked progeny RNA and infectious virus (>107 pfu/ml) in these reactions provides us with an efficient system for genetic and biochemical studies. This cell-free system allows us to investigate most of the molecular events that occur during viral RNA replication in vivo, and it provides us with direct access to the RNA replication complexes which are shielded from experimental manipulation in infected cells. With the availability of infectious cDNA clones for other viruses, it is now possible to adapt this general experimental system to investigate the replication of other picornaviruses and other positive-strand RNA viruses such as bovine viral diarrheal virus (BVDV) and hepatitis C virus (HCV).

By using the in vitro RNA replication system described above, we have been able to study the initiation and elongation of both negative- and positive-strand RNA synthesis. By forming RNA replication complexes in the presence of both mutant and helper RNAs, we have developed an in vitro complementation assays. This assay has allowedus to identify which processed form of a given viral protein is required for a specific step in the RNA replication cycle. In addition, the in vitro complementation assay has provided us with a means of identifying the cis-active sequences in the viral genome. Overall, these findings will have a significant impact on our understanding of molecular mechanisms involved in the replication of positive-strand RNA viruses.

The agenda for biochemical studies in our lab include the identification and characterization of the viral and cellular proteins which are required for the individual steps in the viral RNA replication cycle. We are also examining the role of RNA structure and protein-RNA interactions in the regulating the translation, replication and stability of viral RNAs. Finally, work is now in progress to express and characterize the HCV and the BVDV RNA-dependent RNA polymerases using the HeLa S10 in vitro translation/replication system.

Current genetic studies include the use of site-directed mutagenesis, complementation and recombination assays to characterize the molecular basis of positive-strand RNA virus replication. We are characterizing the cis-active RNA sequencesin the viral genomes and determining how these sequences regulate viral protein synthesis, RNA replication and RNA stability. RNA sequences and structures in 3´and 5'non-translated regions of the viral genome are being investigated to determine how they interact with viral and cellular proteins to form the ribonucleoprotein complexes that are required for the initiation of viral RNA replication.