Inter-domain coordination essential for dengue virus non-structural (5) NS5 interaction with stem loop a (SLA).

Abstract

Dengue virus is the most prevalent arthropod-borne virus and is a part of the flavivirus family. There are four known dengue serotypes, and humans can be infected with more than one serotype which can lead to severe dengue. There are no clinically approved antiviral drugs for the treatment of dengue infection to date, and vaccines available are limited to seropositive individuals. The non-structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is a major component of the viral replication complex. Dengue NS5 has an N-terminal methyltransferase (MTase) domain responsible for 5’ RNA capping, and a C-terminal RNA-dependent-RNA-polymerase (RdRp) domain responsible for de novo RNA synthesis. Dengue NS5 is also known to interact with RNA elements in the 5’-untranslated regions to promote viral RNA synthesis, in particular the 5’ stem loop A (SLA). However, there is limited structural characterization of the dengue NS5/SLA complex. Here, we characterized the interaction of the full length and individual domains of dengue serotype 2 (DENV2) NS5 with SLA at the 5’-UTR using surface plasmon resonance (SPR) studies, electromobility shift assays (EMSAs) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). HDX-MS studies were used to probe the structural dynamics of DENV2 NS5 and its binding interface with SLA. Results from these studies suggest that coordination is required between the MTase and RdRp domains, which stabilize the interaction between NS5 and SLA. Ultimately, our results will shed more light into how dengue NS5 interacts with SLA to discriminate viral RNA from cellular RNA.