Dengue virus (DV), a positive-stranded RNA virus of the Flaviviridae family, is one of the most significant mosquito-borne human viral pathogens. The DV genome consists of a 5'-capped RNA of ~11 kb that encodes three structural proteins and seven non-structural (NS) proteins. DV NS proteins and their interactions are critical for the assembly of an ordered replicase complex for viral RNA replication.
Viral replicons that replicate autonomously and efficiently in cultured cells have been widely used as a tool for the reverse genetic analysis of the function viral protein gene and viral RNA element in viral RNA replication and virion morphogenesis. We have established several DV replicon systems. A selectable DV subgenome replicon was found to be eligible for performing mutagenesis analysis of viral genes and viral RNA elements and for the screening of suppressor that restored the impairment in viral RNA replication of the primary mutation. We are using this DV replicon system to explore the function of DV NS protein genes in DV replication. Mutagenesis analysis of NS protein genes will be performed and inter- and intra-genic suppressors of defective NS protein gene mutations will be screened and verified for their rescuing activity in viral RNA replication. Based on these findings, a model for the arrangement of the cytosolic NS3 and NS5, the endoplasmic reticulum lumen located NS1, and the intracellular membrane anchored NS4A and NS4B in replicase complex could be established; and a genetic interaction network of NS protein genes for functional replicase complex constitution could be built.
We plan to identify the structural/sequence determinants resided in the terminal regions of DV genome that are critical for maintaining a balanced structural inter-conversion between the mutually exclusive linear and circular forms of DV genome. Mutagenesis analysis of the RNA elements that are present in the linear or circular form of DV genome will be performed in the context of a DV selectable subgenome replicon, suppressors for defective mutations will be screened, the impact of primary mutation on RNA structural alteration will be analyzed, and the function of cognate suppressor in restoring a balanced RNA structural inter-conversion for viral RNA replication will be evaluated. Findings of this research may provide a better understanding of the dynamic RNA structural changes in DV life cycle.