Research Summary

Probing Replication by RNA-dependent RNA Polymerases from Flaviviruses

Almost half of the world's population is at risk of infection from Flavivirus family of viruses. For example, the highly prevalent Dengue virus poses a global public health challenge in absence of an effective vaccine or antivirals. After uptake of the virus by the cell and release of the viral genome into the cytoplasm, cognate RNA dependent RNA polymerase (RdRP) must replicate the genomic RNA specifically from a milieu of cellular RNA molecules to create high titers of the new viruses. Moreover, error-prone RdRPs must initiate synthesis correctly to maintain the integrity of the viral genome during exponential genome replication. The replicative complex also must undergo structural and constitutional changes to switch from single-stranded template based negative strand synthesis to double-stranded based positive strand synthesis. Our understanding of how such complex and sometimes contradictory processes are achieved and regulated by various viral and host factors are far from complete.

We will identify the various RdRP intermediates and measure the kinetics of the replication process. The role of the viral RNA in modulating the RdRP activity and form during initiation will be probed. By developing a novel single molecule based methodology, we will circumvent the issues inherent with heterogeneous nature of the RdRP complexes to make precise assignment of the structural forms.

Viral replication is also highly organized temporally and spatially inside the cell and requires interaction of the polymerase-RNA complex with other host/viral factors. Importantly, the exponential phase of the positive strand generation occurs in membrane bound replicase complexes whose constituents and stoichiometries are not known. One putative constituent is the NS3 helicase-protease is essential for positive strand synthesis but its coordination with NS5 protein is not well understood. Using a sheet illumination based live cell super-resolution microscopy, we will probe the stoichiometry of the replicase complexes. Further combined with single molecule FRET imaging in living cells, we will probe the influence of the helicase activity in the replicative processivity and kinetics. Finally, our approach will not only provide insights into the mechanistic details of the replication process but also aid in the diagnostics and drug discovery process by providing novel viral replication assays.

Figure Legend: The Dengue virus genome needs to replicate efficiently to sustain a viable infection cycle. The released genomic RNA is bound and copied by the RNA dependent RNA polymerase to generate the new viral RNA. The proposal will generate insight about various aspects of the dengue virus replication using novel single molecule techniques.