Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5

Abstract

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 ...

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

“…2 2 2 2 2 2 2 DENV2 2 2 2 2 2 2 2 YFV 2 2 2 2 2 2 2 Uninfected 2 2 2 2 2 2 2 staining throughout the cytoplasm with negligible nuclear localization of NS5 (Fig. 1c), consistent with previous observations using polyclonal rabbit antiserum to WNV KUN NS5 (Malet et al, 2007;Mackenzie et al, 2007). Other mAbs in the panel showed similar staining patterns (results not shown).…”

“…All WNV KUN -coding sequences for RdRp domains and the full-length NS5 protein were cloned into the vector pDEST14 using the Gateway system (Life Technologies) with an N-terminal 6His tag. The N-terminally truncated recombinant plasmids used in this study have been described previously (Malet et al, 2007). C-terminal deletion constructs (Fig.…”

“…Protein expression was performed as described previously (Malet et al, 2007). pDEST14 constructs were expressed in Rosetta 2 (DE3) [pLysS] competent cells (Novagen).…”

“…NS5 comprises an N-terminal methyltransferase (MTase) domain that catalyses both guanine N-7 and ribose 29-O methylations during viral RNA cap formation (Zhou et al, 2007) and a C-terminal domain that harbours the RNA-dependent RNA polymerase (RdRp) (Tan et al, 1996;Guyatt et al, 2001;Nomaguchi et al, 2004;Selisko et al, 2006). The crystal structure of the WNV RdRp revealed classic RdRp structure bearing palm, thumb and finger subdomains (Malet et al, 2007).…”

“…Thus, the structure of the full-length molecule is required to provide a more accurate model for functional analysis and drug design. While recent studies have elucidated separate crystal structures for the MTase and RdRp domains of WNV NS5 (Malet et al, 2007;Zhou et al, 2007), apparent instability of the full-length recombinant protein during purification precluded crystallization of the entire molecule. A possible solution to this dilemma is co-purification and co-crystallization of NS5 in the presence of a monoclonal antibody (mAb) that binds to both MTase and RdRp domains and stabilizes the full-length protein.…”