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Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0-and 2.35-Å resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.The Flaviviridae form a large family of single-stranded positive-sense RNA viruses comprising the three genera Hepacivirus, Pestivirus, and Flavivirus. The genus Flavivirus contains more than 80 known arthropod-borne viruses, including major human and animal pathogens such as dengue virus (DENV), 3 yellow fever virus, Japanese encephalitis virus, and West Nile virus (WNV). Both DENV and WNV are considered as emerging pathogens. Dengue fever is one of the most important mosquito-borne viral diseases in the world, with more than 3 billion people at risk in endemic tropical areas (1). Dengue outbreaks are increasingly severe in terms of cases and fatalities in many regions of the world (2). WNV was discovered in the West Nile district in Uganda in 1937 and was subsequently shown to have an extensive worldwide distribution with the exception of the Americas (1). In 1999, WNV was introduced into the Americas in the New York City area and has since spread throughout the mainland United States, southern Canada, and Mexico. WNV epidemics in the United States have resulted in a total of 23,925 cases of human disease and 946 deaths reported to the Centers for Disease Control (CDC) from 1999 to 2006. WNV consists of 2 lineages (I and II). The North American WNV isolates belong to lineage I, which also includes the Australian subtype Kunjin (3). In contrast to other lineage I WNV strains (4), infections with the Kunjin subtype of WNV do not cause fatal disease in humans (5).The Flavivirus positive sense RNA genome contains a single open reading frame encoding a polyprotein that is processed into three structural and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Signature-sequence analysis suggests that the ...

†These authors contributed equally to this publication.Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin a/b and importin b1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartitetype aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K 387 K 388 K 389 ); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the Nterminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R 376 KNGK 380 , abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R 184 KR 186 and D 436 GEE 439 , we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D 334 EE 336 and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.The four serotypes of Dengue virus (types 1 to 4) belong to the family Flaviviridae, which consists of the genera Flavivirus, Pestivirus, and Hepacivirus (52). The dengue virus genome is positive-sense RNA of 11 kb and encodes the proteins C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 in a single open reading frame. Co-and posttranslational polyprotein processing by host and viral proteinases generates three structural proteins, namely, C (capsid), M (membrane associated) and E (envelope), and seven nonstructural (NS) proteins, NS1 through NS5 (reviewed in reference 45). Biochemical functions have been demonstrated for some nonstructural proteins. NS5 possesses RNA-dependent RNA polymerase activity (49). A complex of NS2B and NS3 acts as a chymotrypsin-like serine proteinase; the N-terminal 30% of NS3 is sufficient for this activity (15, 42). The C-terminal 70% of NS3 has seven motifs characteristic of RNA helicases of the DExH subfamily. Recombinant proteins containing the C-terminal helicase region of dengue virus NS3 possess n...