Mechanism of Rotavirus Genome Replication and Packaging

Rotaviruses are the most important cause of severe dehydrating diarrhea in infants and young children. The mortality due to rotavirus infection is significant, globally causing nearly 1 million deaths each year. Recently, a vaccine was developed in the Laboratory of Infectious Diseases that provided a high level of protection against the severe diarrhea and dehydration associated with rotavirus infection. Because of concerns that the vaccine may infrequently cause intussusception in young children, the vaccine was withdrawn from the US market by the licensee Wyeth-Lederle. Due to the impact of rotavirus infection on human health, an important overall goal of the Laboratory remains the development of new vaccines, the improvement of existing vaccines, and the identification of other methods for preventing and treating rotaviral disease. Accomplishing this goal would be helped by a fuller understanding of the molecular biology of the virus. In particular, by defining events in the replication and packaging of the segmented double-stranded RNA genome of these viruses, we should gain the information necessary to attenuate pathogenic strains of rotavirus and thereby to molecularly engineer new candidate vaccines. The primary focus of this project is to characterize the fundamental events in the replication of the rotavirus genome which then can be used to develop a reverse genetics system for manipulating the genetic information of the virus. Specifically, this project seeks to identify cis-acting signals that are important for the transport, sorting, packaging and replication of rotavirus mRNAs. The project also seeks to characterize the structure and function of those viral proteins involved in these processes. These aims will be accomplished by a combination of procedures, which include (i) analysis of the replication and translation efficiencies of mutated viral mRNAs with cell-free systems, (ii) computer modeling and structural analysis (e.g., RNAse mapping, NMR spectroscopy) of the recognition signals in the mRNAs, (iii) characterization of the enzymatic and structural properties (e.g., analytical ultracentrifugation, X-ray crystallography, cryo-electron microscopy, CD spectroscopy) of recombinant viral proteins, and (iv) elucidation of the specificity and targets of the viral RNA-binding proteins by gel mobility shift assay and RNA-protein cross-linking. Studies performed in the last year have provided insight into (i) the requirements for initiation of minus strand synthesis by the viral RNA polymerase, (ii) the nature of cis-acting signals in viral mRNAs that promote the synthesis of the dsRNA genome, (iii) the enzymatic activities of the potential RNA packaging protein NSP2, and (iv) the identity of the viral protein responsible for methylation of the 5 prime-caps of rotavirus mRNAs.