Structural & functional characterization of the dengue virus non-structural protein 5 (NS5)

Abstract

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 multifunctional 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 catalyticallycompetent NS5-SAH-cap-0-viral RNA methylation complex, mutagenesis studies targeting the Abstract xvi 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 rathe...