The Two-component NS2B-NS3 Proteinase Represses DNA Unwinding Activity of the West Nile Virus NS3 Helicase

Andrei V Chernov,Sergey A Shiryaev,Alexander E Aleshin,Boris I Ratnikov,Jeffrey W Smith,Robert C Liddington,Alex Y Strongin

doi:10.1074/jbc.m801719200

Abstract

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.

Highlights

Specific therapy available for any flavivirus infection
The hydrophilic 48-amino acid central part of the natural membrane-tethered NS2B cofactor was linked via a flexible GGGGSGGGG linker to the 1505–2123 sequence of the West Nile virus (WNV) polyprotein precursor, which encodes NS3pro-hel
Because of the cleavage at GER2MDE173, the 51-kDa species commenced from Met171 and represented the individual NS3 helicase domain (NS3hel) domain

Summary

Introduction

Specific therapy available for any flavivirus infection. The discovery of potent therapeutic compounds against the medically important flaviviruses is urgently needed. Similar to many flavivirus types including Kunjin, DV, yellow fever virus, and Japanese encephalitis virus, the full-length NS3 peptide sequence of WNV represents a multifunctional protein in which the N-terminal 184 residues encode serine proteinase (NS3pro) and the C-terminal 440 residues code for an RNA triphosphatase, an NTPase, and an RNA helicase (NS3hel) [4]. The folding and the spatial structure of the NS3 proteinase domain alone are significantly different from those of the two-component NS2B-NS3 proteinase complex [14, 15, 17, 18] Because both the plus and minus strands of template RNA are highly structured, the viral replicating machinery requires the unwinding of the RNA secondary structure in the template RNAs. Because both the plus and minus strands of template RNA are highly structured, the viral replicating machinery requires the unwinding of the RNA secondary structure in the template RNAs This important function is performed by NS3hel. Flaviviruses with impaired NS3pro or NS3hel or both are unable to replicate, making the NS3pro-hel biological system a promising drug target for antiviral therapy [21,22,23,24,25,26,27]

Methods

Results

Conclusion