Viruses from the Flavivirus family are the causative agents of dengue fever, Zika, Japanese encephalitis, West Nile encephalitis or Yellow fever and constitute major or emerging public health problems. A better understanding of the flavivirus replication cycle is likely to offer new opportunities for the design of antiviral therapies to treat severe conditions provoked by these viruses, but it should also help reveal fundamental biological mechanisms of the host cell. During virus replication, RNA synthesis is mediated by a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC). The RC is composed of both viral and host-cell proteins that assemble within vesicles composed of the endoplasmic reticulum membrane, near the nucleus. At the heart of the flavivirus RC lies NS4B, a viral integral membrane protein that plays a role in virulence and in down-regulating the innate immune response. NS4B binds to the NS2B-NS3 protease-helicase, which itself interacts with the NS5 methyl-transferase polymerase. We present an overview of recent structural and functional data that augment our understanding of how viral RNA is replicated by dengue virus. We focus on structural data that illuminate the various roles played by proteins NS2B-NS3, NS4B and NS5. By participating in viral RNA cap methylation, the NS5 methyltransferase enables the virus to escape the host cell innate immune response. We present the molecular basis for this activity. We summarize what we know about the network of interactions established by NS2B-NS3, NS4B and NS5 (their "interactome"). This leads to a working model that is captured in the form of a rather naïve "cartoon", which we hope will be refined towards an atomic model in the near future.

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...