(793.10) Insights into the Binding of the Dengue Virus Nonstructural 5 (NS5) Protein to Stem Loop A (SLA)

Poster Board Number: A124

Juliet Obi (University of Maryland), James Fields (University of Maryland), Linfah McQueen (University of Maryland), Daniel Deredge (University of Maryland)

Dengue virus (DENV) is the most prevalent mosquito-borne virus that infects humans, and it continues to pose a public health threat to over 2.5 billion people globally. There are no clinically approved antivirals available to treat dengue infection to date, and available vaccines are limited to seropositive individuals which calls for intensified research efforts in therapeutic development. The non-structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is responsible for viral RNA genome replication. 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. These two domains are connected by a flexible linker which allows NS5 to adopt multiple conformations in performing its multiple replicative functions. The dual enzymatic activity of NS5 and its essential role in dengue viral RNA replication, makes it an attractive target for the development of antiviral for the treatment of dengue infection. However, there is limited understanding on the structural basis for the interactions of NS5 to its different RNA partners during the viral replication cycle. Studies have shown that dengue NS5 discriminates between viral and host RNA by binding to RNA elements as the 5’-untranslated regions (5’-UTR) including stem loop A (SLA) to promote viral RNA synthesis. In this study, we characterized the binding of NS5 from dengue virus serotype 2 (DENV2) with stem loop A (SLA) at the 5’-UTR using surface plasmon resonance (SPR) studies and determined their binding stoichiometry using analytical ultracentrifugation (AUC) studies. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) studies of DENV2 NS5 alone and in complex with SLA reveal large conformational fluctuations consistent with an EX1 kinetic regime of HDX-MS at the interface between the MTase and RdRp domains and identify regions of interactions between DENV2 NS5 and SLA. These experiments were repeated in the context of the individual MTase or RdRp domains to gain insight on how they coordinate binding to SLA compared to full-length NS5. The results from our study will advance our understanding of how dengue NS5 binds SLA and its other RNA interaction partners.

This work is supported partially by the University of Maryland, Baltimore School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014), and by NIG-NIGMS grant T32 GM066706 (J.O.O).