Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts.

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex

ACS Infectious Diseases ◽

10.1021/acsinfecdis.0c00554 ◽

2020 ◽

Vol 6 (10) ◽

pp. 2800-2811

Author(s):

Anand Balakrishnan ◽

Edmund Price ◽

Catherine Luu ◽

Jacob Shaul ◽

Charles Wartchow ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rna Polymerase ◽

Biochemical Characterization ◽

Rna Dependent Rna Polymerase ◽

Polymerase Complex ◽

Virus Rna ◽

Syncytial Virus

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Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase

RNA ◽

10.1261/rna.031914.111 ◽

2012 ◽

Vol 18 (8) ◽

pp. 1541-1552 ◽

Cited By ~ 9

Author(s):

R. Vaughan ◽

B. Fan ◽

J.-S. You ◽

C. C. Kao

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Functional Characterization ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna ◽

Nascent Rna

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West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis

Journal of General Virology ◽

10.1099/vir.0.82552-0 ◽

2007 ◽

Vol 88 (4) ◽

pp. 1163-1168 ◽

Cited By ~ 46

Author(s):

Jason M. Mackenzie ◽

Mark T. Kenney ◽

Edwin G. Westaway

Keyword(s):

West Nile Virus ◽

Rna Polymerase ◽

Virus Strain ◽

Viral Rna ◽

West Nile ◽

Replication Complex ◽

Virus Rna ◽

West Nile Virus Strain ◽

First Time ◽

Cytoplasmic Location

Using West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.

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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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