Virtually nothing is known of the mechanism of replication of double- stranded (ds) genome RNA for members of the Reoviridae. Recently, tempIate-dependent, in vitro replication systems have been generated for dsRNA viruses of yeast and bacteria, and rapid advances have been made in those systems. Here, describe preliminary results obtained with a template-dependent, in vitro replication system derived from rotavirus. This system utilizes a particulate replicase activity and messenger- sense, single-stranded, template RNAs of rotavirus origin, and initiates and completes synthesis of authentic rotavirus dsRNA. Both the particulate replicase and template components of the in vitro replication system have been derived from cloned and expressed rotavirus genes, making all components of the system amenable to molecular manipulation. In this proposal, I present four specific aims that exploit this template-dependent, in vitro replication system for rotaviruses. The first three specific aims are directed at identifying signals on templates and domains within proteins of the particulate replicase that regulate the replication of rotavirus RNA. The fourth specific aim is directed at exploiting the system to achieve rescue of cloned and manipulated rotavirus genes into infectious virus with the goal of opening rotaviruses to """"""""reverse genetics."""""""" In Aim one I will biochemically optimize the in vitro replication system, characterize the mode and site of initiation of (-)-strand synthesis, explore the possibility that the number of components in the particulate replicase can be reduced, and develop a series of reporter template RNAs for use in the system. Experiments in Aim two will identify the cis- acting signals present on the template RNAs that regulate their replication. Whether there are template site-filling requirements for replicase activation, and whether foreign RNAs can be replicated, will also be determined.
Aim three proposes to define a binding assay to detect specific recognition and binding of template to the replicase. This assay will be used to identify cis-acting binding sites on the template RNAs and replicase protein components, and domains within those proteins, that interact with those template binding sites.
In Aim four the in vitro replication system will be applied to the problem of rescue of cloned and manipulated rotavirus genes into infectious virus. The techniques of PCR-cloning, DNA sequencing, deletion mapping, site- directed mutagenesis, and baculovirus-expression of rotavirus gene products will be used extensively in the proposed experiments.
| Wentz, M J; Patton, J T; Ramig, R F (1996) The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis. J Virol 70:7833-41 |
| Zeng, C Q; Wentz, M J; Cohen, J et al. (1996) Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol 70:2736-42 |
| Wentz, M J; Zeng, C Q; Patton, J T et al. (1996) Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro. Arch Virol Suppl 12:59-67 |
