Structural & functional characterization of the dengue virus non-structural protein 5 (NS5)
Soh, Sherryl Tingjin
Date of Issue2018
School of Biological Sciences
Dengue virus (DENV) is the most important arthropod-borne pathogens capable of causing human mortality and morbidity. Currently, there are no antiviral drugs available for treatment of dengue infections. Although a tetravalent DENV vaccine has recently been licensed for use, it has limited efficacy. For DENV, NS5 is the best characterized and most conserved multi-functional protein comprising an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp). Both play essential roles in viral replication in the host cell. The crystal structure of the DENV full-length NS5 revealed a well-ordered linker region and an inter-domain interface mostly formed by polar residues. Using a combination of biochemical and reverse genetic approaches, the biological relevance of the flexible linker between MTase and RdRp in the DENV-3 NS5 FL and their intra-molecular interactions was investigated. Several conserved interface residues were shown to be important for viral replication, through influencing either MTase or RdRp activities. Other NS5 alanine mutants displayed comparable enzymatic activities as wild-type, but were either less competent or lethal for virus production, suggesting that they play vital but non-enzymatic roles in viral replication and infectivity. Alanine mutations of the linker region showed that the third and fourth residues of the short 310-helix regulate polymerase de novo initiation activity for viral replication in cells. In addition, linker swapping experiment demonstrated that the unique amino acid composition of the linker controls NS5 conformation flexibility for cross-talk between the two domains and for interaction with viral and host proteins in a serotype/virus-specific manner. By solving crystal structures of ternary complexes between DENV-3 NS5 protein, an authentic cap-0-viral RNA substrate, S-adenosyl-L-homocysteine (SAH) and/or RdRp allosteric inhibitors, we functionally probed these inhibitor and substrate binding sites in the RdRp and MTase with biochemical, biophysical and reverse genetic tools. Based on the catalytically-competent NS5-SAH-cap-0-viral RNA methylation complex, mutagenesis studies targeting the highly conserved capped-RNA binding groove in the MTase domain was performed. The importance of the polar interaction between NS5 residue E111 and G2 base of RNA for viral replication as well as the positional requirement G2 for virus growth were identified. Moreover, residues lining the RNA binding groove exhibited differential reduction in 2’-O methylation activity, indicating that these residues are critical for capped-RNA binding and 2’-O methyl transfer reaction. Using compound and fragment-based screening coupled with structure-guided design, we identified two classes of allosteric inhibitors that bound either to the F1 motif, or to the thumb subdomain and priming loop (termed “N-pocket”) of the DENV RdRp. Antiviral activities of F1 motif and N-pocket inhibitors were primarily due to an impact on polymerase de novo initiation activity rather than elongation during RNA synthesis. Additionally, kinetic characterization showed that the N-pocket inhibitors exhibited mixed inhibition profiles when compete against the RNA or GTP substrate. Resistant mutants raised from these inhibitors were also mapped to the N-pocket of RdRp, confirming that they bind specifically to this pocket to block viral replication. The proposed mode of action for N-pocket compounds is to prevent NS5 RdRp de novo initiation and block conformation changes during transition from initiation to elongation. In order to examine how the viral RNA is recognized and replicated as well as to facilitate drug discovery and design targeting the RdRp, we attempted to obtain crystal structure of NS5 RdRp bound to RNA. A novel fluorescence polarization (FP)-based assay was developed to profile various distinct RNA constructs for their suitability in co-crystallization. Several RNA substrates demonstrated good binding affinity to NS5 protein and were capable of forming functional elongation complexes. Crystallization trials using commercial screening kits were set up, but no crystal structure with bound RNA was obtained. Future work will aim at optimizing the conditions during assembly and reaction in order to attain more soluble and stable elongation complexes for crystallization. Overall, these findings provide valuable information on the functions and dynamics of NS5 as well as its molecular interactions with substrates and inhibitors, and have significant implications for the development of antiviral drugs targeting flaviviruses.