scholarly journals Identification of Residues Critical for the Interferon Antagonist Function of Langat Virus NS5 Reveals a Role for the RNA-Dependent RNA Polymerase Domain

2007 ◽  
Vol 81 (13) ◽  
pp. 6936-6946 ◽  
Author(s):  
Gregory S. Park ◽  
Keely L. Morris ◽  
Roselyn G. Hallett ◽  
Marshall E. Bloom ◽  
Sonja M. Best
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Janus Kinase ◽  
Amino Acid Residues ◽  
Rna Dependent Rna Polymerase ◽  
Stat Signaling ◽  
Langat Virus ◽  
Tick Borne Encephalitis Virus

ABSTRACT All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.

scholarly journals An RNA-dependent RNA polymerase inhibitor for tick-borne encephalitis virus

Virology ◽  
2020 ◽  
Vol 546 ◽  
pp. 13-19
Author(s):  
Varun Gejji ◽  
Pavel Svoboda ◽  
Michal Stefanik ◽  
Haoqi Wang ◽  
Jiri Salat ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Encephalitis Virus ◽  
Rna Dependent Rna Polymerase ◽  
Tick Borne Encephalitis ◽  
Polymerase Inhibitor ◽  
Tick Borne Encephalitis Virus

scholarly journals Blocking of Interferon-Induced Jak-Stat Signaling by Japanese Encephalitis Virus NS5 through a Protein Tyrosine Phosphatase-Mediated Mechanism

2006 ◽  
Vol 80 (12) ◽  
pp. 5908-5918 ◽  
Author(s):  
Ren-Jye Lin ◽  
Bi-Lan Chang ◽  
Han-Pang Yu ◽  
Ching-Len Liao ◽  
Yi-Ling Lin
Keyword(s):  
Japanese Encephalitis Virus ◽  
Tyrosine Phosphorylation ◽  
Japanese Encephalitis ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Encephalomyocarditis Virus ◽  
Janus Kinase ◽  
Protein Tyrosine ◽  
Stat Signaling

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes severe human disease, has been shown to block the interferon (IFN)-induced Janus kinase signal transducer and activation of transcription (Jak-Stat) signaling cascade by preventing Tyk2 tyrosine phosphorylation and Stat activation. In this study, we demonstrate that expression of the JEV nonstructural protein NS5 readily blocked IFN-stimulated Jak-Stat signaling events such as Stat1 nuclear translocation and tyrosine phosphorylation of Tyk2 and Stat1. The region of JEV NS5 responsible for Stat1 suppression was identified using various deletion clones. Deletion of 83 N-terminal residues of JEV NS5, but not the 143 C-terminal residues, abolished its ability to block IFN-stimulated Stat1 activation. The role of JEV NS5 as an IFN antagonist was further demonstrated by its ability to block the induction of interferon-stimulated genes and the antiviral effect of IFN-α against the IFN-sensitive encephalomyocarditis virus, which appears to replicate and kill cells that express NS5 even with alpha IFN treatment. Furthermore, the molecular mechanism responsible for IFN antagonism by NS5 probably involves protein tyrosine phosphatases (PTPs), as the IFN-blocking events in both JEV-infected and NS5-expressing cells were reversed by sodium orthovanadate, a broad-spectrum inhibitor of PTPs. We suggest that JEV NS5 is an IFN antagonist and that it may play a role in blocking IFN-stimulated Jak-Stat signaling via activation of PTPs during JEV infection.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

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.

Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits

2016 ◽  
Vol 50 (2) ◽  
pp. 307-312
Author(s):  
Y. V. Kuzmenko ◽  
E. S. Starodubova ◽  
G. G. Karganova ◽  
A. V. Timofeev ◽  
V. L. Karpov
Keyword(s):  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Nonstructural Protein 1 ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus

2008 ◽  
Vol 82 (17) ◽  
pp. 8272-8282 ◽  
Author(s):  
Wolfgang Fischl ◽  
Sigrid Elshuber ◽  
Sabrina Schrauf ◽  
Christian W. Mandl
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Encephalitis Virus ◽  
Wild Type Virus ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Tick Borne Encephalitis ◽  
Protease Specificity ◽  
Tick Borne Encephalitis Virus ◽  
Cleavage Motif

ABSTRACT The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.

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

2013 ◽  
Vol 94 (9) ◽  
pp. 1961-1971 ◽  
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.

scholarly journals Proteinase-Polymerase Precursor as the Active Form of Feline Calicivirus RNA-Dependent RNA Polymerase

2001 ◽  
Vol 75 (3) ◽  
pp. 1211-1219 ◽  
Author(s):  
Lai Wei ◽  
Jason S. Huhn ◽  
Aaron Mory ◽  
Harsh B. Pathak ◽  
Stanislav V. Sosnovtsev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Active Form ◽  
Polymerase Activity ◽  
Feline Calicivirus ◽  
Amino Terminus ◽  
Rna Dependent Rna Polymerase ◽  
Reading Frame ◽  
Catalytically Active ◽  
Catalytic Cysteine

ABSTRACT The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

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