Virus replication is achieved through the precise coordination of a series of macromolecular synthetic events. For plus-strand RNA viruses the temporal regulation of gene expression and genome replication occurs through the action of the viral ribonuclear protein complex (vRNP). The vRNP is assembled around viral RNA and its composition is defined by the recruitment of proteins, both viral and host, to specific RNA elements. Differential availability of proteins and viral RNA elements at particular times during the virus replication cycle can therefore alter the composition of the vRNP and change the function of the complex. Identifying RNA elements required for specific macromolecular synthetic events and the proteins that bind them to form a vRNP are fundamental requirements if we are to understand the regulatory processes necessary for virus replication.
This project seeks to understand the regulation of gene expression and genome replication of plus-strand RNA viruses using Sindbis virus (SIN), the type species of the alphavirus genus. During SIN replication the genome is translated giving rise to the non-structural polyprotein. A semi-processed form of this protein is required for the copying (minus-strand synthesis) of the genome from which it was translated. In turn the minus-strand RNA serves as a template for production of progeny genomes, and a subgenomic mRNA synthesized by a complex containing the fully processed non-structural proteins. It is therefore apparent that different vRNP complexes perform each of these processes. The project aims to elucidate the molecular interactions that mediate the transitions in vRNP composition and function
These studies will significantly impact the understanding of the molecular mechanisms required for alphavirus replication. Additionally, as plus-strand RNA viruses are united by a common scheme of replication, principles established from these studies will be of more general interest and importance for the study of all plus-strand RNA viruses. The project will provide a forum for the training of undergraduate students, graduate students, and post-doctoral fellows. A number of students from various programs (including Initiative for Maximizing Student Diversity Program and Research Experience for Undergraduates Program) have participated in research in the laboratory and it is fully expected that this participation will continue.
Alphaviruses are mosquito-borne pathogens of humans and livestock and a significant cause of morbidity and mortality world-wide. Sindbis virus is the type species of the alphavirus genus and represents an excellent model system in which to analyze requirements for efficient virus replication. The aims of this project were to understand how specific regions of the viral genome functioned to promote viral replication, examine functions of the viral polymerase, and gain a greater understanding of the interaction of the virus with specific components of the host cell. Over the course of this award we have published a manuscript that identifies a structural element at the 5' end of the viral genome as essential for virus replication. An extension of that work has gone on to show that the stability of that structure is important for function. If the structure is too stable the virus synthesizes RNA less well and grows slower and to lower titers. We have also been able to purify the viral RNA polymerase. This was a significant breakthrough that has allowed clean analyses of the activities associated with this protein. We have shown that the polymerase is not only capable of copying viral RNA, but also able to polyadenylate RNA. Detailed mutational analyses of the polymerase also identified the amino-terminal domain as being important for RNA synthesis and results we published suggest that this region makes multiple contacts with other viral proteins that are necessary for RNA synthesis at different points during the viral replication cycle. More recent work has shown that this region of the polymerase assists in the appropriate cellular localization of other viral proteins and that when this localization is disrupted host cell gene expression is not inhibited and viral replication is reduced. These later findings are beginning to elucidate interactions between the virus and host cell that are essential for efficient virus replication and may provide insight into the means by which the virus controls host gene expression during infection.
