scholarly journals Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate

2006 ◽  
Vol 398 (3) ◽  
pp. 557-567 ◽  
Author(s):  
Isabelle Bougie ◽  
Martin Bisaillon
Keyword(s):  
Fluorescence Spectroscopy ◽  
Competitive Inhibitor ◽  
Viral Mrnas ◽  
Dna Viruses ◽  
Chlorella Virus ◽  
Virus Rna ◽  
Rna Triphosphatase ◽  
Rna Triphosphatases ◽  
Cd Studies ◽  
Structural Indicators

Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5′ end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.

scholarly journals RNA Triphosphatase Component of the mRNA Capping Apparatus of Paramecium bursaria Chlorella Virus 1

2001 ◽  
Vol 75 (4) ◽  
pp. 1744-1750 ◽  
Author(s):  
C. Kiong Ho ◽  
Chunling Gong ◽  
Stewart Shuman
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Initial Step ◽  
Genetic System ◽  
Paramecium Bursaria ◽  
Viral Mrnas ◽  
Dna Viruses ◽  
Rna Triphosphatase ◽  
Rna Triphosphatases

ABSTRACT Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5′ diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the γ-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show thatChlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

scholarly journals RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

2009 ◽  
Vol 90 (7) ◽  
pp. 1748-1756 ◽  
Author(s):  
M. Gopinath ◽  
M. S. Shaila
Keyword(s):  
Biochemical Characterization ◽  
Covalent Bond ◽  
Rinderpest Virus ◽  
Viral Mrnas ◽  
L Protein ◽  
Rna Triphosphatase ◽  
Rnp Complex

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of γ-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

scholarly journals Energetics of RNA binding by the West Nile virus RNA triphosphatase

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 867-877 ◽  
Author(s):  
Ines Benzaghou ◽  
Isabelle Bougie ◽  
Frédéric Picard-Jean ◽  
Martin Bisaillon
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
The West ◽  
West Nile ◽  
Virus Rna ◽  
Rna Triphosphatase

Regulation of translation initiation by herpesviruses

2008 ◽  
Vol 36 (4) ◽  
pp. 701-707 ◽  
Author(s):  
Richard W.P. Smith ◽  
Sheila V. Graham ◽  
Nicola K. Gray
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Translational Regulation ◽  
Large Family ◽  
Cell Protein ◽  
Viral Protein Synthesis ◽  
Viral Mrnas ◽  
Dna Viruses ◽  
Protein Synthesis Machinery ◽  
Regulate Protein

Viruses are dependent upon the host cell protein synthesis machinery, thus they have developed a range of strategies to manipulate host translation to favour viral protein synthesis. Consequently, the study of viral translation has been a powerful tool for illuminating many aspects of cellular translational control. Although much work to date has focused on translational regulation by RNA viruses, DNA viruses have also evolved complex mechanisms to regulate protein synthesis. Here we summarize work on a large family of DNA viruses, the Herpesviridae, which have evolved mechanisms to sustain efficient cap-dependent translation and to regulate the translation of specific viral mRNAs.

scholarly journals Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

2008 ◽  
Vol 82 (11) ◽  
pp. 5573-5583 ◽  
Author(s):  
Yi Li ◽  
Linda A. Guarino
Keyword(s):  
Double Mutant ◽  
Essential Gene ◽  
Direct Analysis ◽  
Mutant Virus ◽  
Late Gene Expression ◽  
Rna Triphosphatase ◽  
Capping Enzyme ◽  
Rna Triphosphatases ◽  
In The Wild ◽  
Budded Virus

ABSTRACT The baculovirus Autographa californica nucleopolyhedrovirus encodes two proteins with RNA triphosphatase activity. Late expression factor LEF-4, which is an essential gene, is a component of the RNA polymerase and also encodes the RNA capping enzyme guanylyltransferase. PTP/BVP is also an RNA triphosphatase, but is not essential for viral replication, possibly because its activity is redundant to that of LEF-4. To elucidate the role of these proteins in mRNA cap formation, a mutant virus that lacked both RNA triphosphatase activities was constructed. Infection studies revealed that the double-mutant virus was viable and normal with respect to the production of budded virus. Pulse-labeling studies and immunoblot analyses showed that late gene expression in the double mutant was equivalent to that in the wild type, while polyhedrin expression was slightly reduced. Direct analysis of the mRNA cap structure indicated no alteration of cap processing in the double mutant. Together, these results reveal that baculoviruses replicate and express their late genes at normal levels in the absence of its two different types of RNA triphosphatases.

scholarly journals The Mechanism of Action of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, a Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

2007 ◽  
Vol 52 (2) ◽  
pp. 458-464 ◽  
Author(s):  
Eisuke Murakami ◽  
Congrong Niu ◽  
Haiying Bao ◽  
Holly M. Micolochick Steuer ◽  
Tony Whitaker ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Potent Inhibitor ◽  
Nucleoside Diphosphate Kinase ◽  
Competitive Inhibitor ◽  
Hcv Rna ◽  
Hepatitis C Virus Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5′-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5′-triphosphate of the uridine congener, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5′-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

scholarly journals Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5′-end of the RNA substrate

2020 ◽  
Vol 295 (27) ◽  
pp. 9076-9086
Author(s):  
Yuko Takagi ◽  
Naoyuki Kuwabara ◽  
Truong Tat Dang ◽  
Koji Furukawa ◽  
C. Kiong Ho
Keyword(s):  
Trypanosoma Cruzi ◽  
Binding Site ◽  
Crystal Structures ◽  
Active Site ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Active Form ◽  
Valuable Insight ◽  
Rna Triphosphatase ◽  
Rna Triphosphatases

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20–2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5′-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

scholarly journals From A to m6A: The Emerging Viral Epitranscriptome

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1049
Author(s):  
Belinda Baquero-Perez ◽  
Daryl Geers ◽  
Juana Díez
Keyword(s):  
Nuclear Export ◽  
Current Knowledge ◽  
Rna Modification ◽  
Structure Stability ◽  
Rna Modifications ◽  
Viral Mrnas ◽  
Dna Viruses ◽  
Mrna Structure ◽  
Cellular Mrnas ◽  
Rna And Dna

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N6-methyladenosine (m6A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, translation and nuclear export. This modification is also found in viral RNA genomes and in viral mRNAs derived from both RNA and DNA viruses. A growing body of evidence indicates that m6A modifications play important roles in regulating viral replication by interacting with the cellular m6A machinery. In this review, we will exhaustively detail the current knowledge on m6A modification, with an emphasis on its function in virus biology.

scholarly journals Mapping the active site of vaccinia virus RNA triphosphatase

Virology ◽  
2003 ◽  
Vol 309 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Chunling Gong ◽  
Stewart Shuman
Keyword(s):  
Vaccinia Virus ◽  
Active Site ◽  
Virus Rna ◽  
Rna Triphosphatase
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