Abstract
BackgroundJapanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.ResultsTo characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template.ConclusionAs a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.
Highlights
Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains
JEV is a member of Flaviviridae family, which consists of the genera Flavivirus (JEV, dengue virus [DEN], yellow fever virus [YF], West Nile virus [WNV], Kunjin virus [KUN], Murray Valley encephalitis virus), Pestivirus, and Hepacivirus
Expression and purification of recombinant JEV NS5 protein To investigate the biochemical properties of JEV RdRp, recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus was expressed in E. coli
Summary
Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. JEV is an enveloped, positive-stranded RNA virus whose genome consists of a single-stranded RNA molecule of approximately 11 kb. The RNA genome of JEV consists of 98-nucleotide (nt) long 5' untranslated region (UTR) with the type-1 cap structure at its 5' terminus, a single open reading frame (ORF), and a 585-nt long 3'-UTR with no poly(A) tail at its 3' terminus [4]. Analysis of the amino acid sequence of NS5 led to the prediction that it carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. The C-terminal region of NS5, which contains the conserved RdRp motifs [8,9] and the Gly-Asp-Asp (GDD) motif found in the active site of many viral RdRps [10], is likely responsible for the RNA polymerase activity
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