Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

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An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Journal of General Virology ◽

10.1099/vir.0.054395-0 ◽

2013 ◽

Vol 94 (9) ◽

pp. 1961-1971 ◽

Cited By ~ 14

Author(s):

Cindy S. E. Tan ◽

Jody M. Hobson-Peters ◽

Martin J. Stoermer ◽

David P. Fairlie ◽

Alexander A. Khromykh ◽

...

Keyword(s):

West Nile Virus ◽

Rna Polymerase ◽

Genetic Interaction ◽

Nonstructural Protein ◽

Specific Interaction ◽

Simian Virus 40 ◽

T Antigen ◽

Rna Dependent Rna Polymerase ◽

West Nile

The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46–49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase–POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase–POL interactions.

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Mutagenesis of the West Nile virus NS2B cofactor domain reveals two regions essential for protease activity

Journal of General Virology ◽

10.1099/vir.0.83447-0 ◽

2008 ◽

Vol 89 (4) ◽

pp. 1010-1014 ◽

Cited By ~ 38

Author(s):

Keith J. Chappell ◽

Martin J. Stoermer ◽

David P. Fairlie ◽

Paul R. Young

Keyword(s):

West Nile Virus ◽

Proteolytic Activity ◽

The West ◽

West Nile ◽

Mutagenesis Screen ◽

Antiviral Compounds ◽

Protease Activation ◽

Primary Focus ◽

Ns3 Protease

The flavivirus NS2B/NS3 protease has received considerable attention as a target for the development of antiviral compounds. While substrate based inhibitors have been the primary focus to date, an approach focussing on NS2B cofactor displacement could prove to be an effective alternative. To understand better the role of the NS2B cofactor in protease activation, we conducted an alanine mutagenesis screen throughout the 42-residue central cofactor domain (NS2B51–92) of West Nile virus (WNV). Two sites critical for proteolytic activity were identified (NS2B59–62 and NS2B75–87), where the majority of substitutions were found to significantly decrease proteolytic activity of a recombinant WNV NS2B/NS3 protease. These findings provide mechanistic insights into the structural and functional role that the cofactor may play in the substrate-bound and free protease complexes as well as providing novel sites for targeting new antiviral inhibitors.

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