Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.
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PMID:Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling. 1804 58

RNA viruses employ a combination of mechanisms to regulate their gene expression and replication. Brome mosaic virus (BMV) is a tripartite positive-strand RNA virus used to study the requirements for virus infection. BMV genomic RNA1 encodes protein 1a, which contains a methyltransferase (MT) domain and a helicase domain that are required for replication. 1a forms a complex with the 2a RNA-dependent RNA polymerase for the replication and transcription of all BMV RNAs. RNA1 expressed with 2a from Agrobacterium-based vectors can result in RNA1 replication in Nicotiana benthamiana. A mutation in the 1a translation initiation codon significantly decreased RNA1 accumulation even when wild-type (WT) 1a and 2a were provided in trans. Therefore, efficient RNA1 replication requires 1a translation from RNA1 in cis, indicating a linkage between replication and translation. Mutation analyses showed that the full-length 1a protein was required for efficient RNA1 replication, not just the process of translation. Three RNA1s with mutations in the 1a MT domain could be partially rescued by WT 1a expressed in trans, indicating that the cis-acting function of 1a was retained. Furthermore, an RNA motif in the 5'-untranslated region of RNA1, named the B box, was required for 1a to function in cis and in trans for BMV RNA accumulation. The B box is required for the formation of the replication factory (M. Schwartz, J. Chen, M. Janda, M. Sullivan, J. den Boon, and P. Ahlquist, Mol. Cell 9:505-514, 2002). Results in this work demonstrate a linkage between BMV RNA1 translation and replication.
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PMID:cis- and trans-acting functions of brome mosaic virus protein 1a in genomic RNA1 replication. 1816 Apr 34

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.
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PMID:Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene. 1819 Sep 94

The maintenance of centromeric heterochromatin in fission yeast relies on the RNA interference-dependent complexes RITS (RNA-induced transcriptional silencing complex) and RDRC (RNA-directed RNA polymerase complex), which cooperate in a positive feedback loop to recruit high levels of histone H3 K9 methyltransferase activity to centromeres and to promote the assembly and maintenance of centromeric heterochromatin. However, it is unclear how these complexes are targeted to chromatin. RITS comprises Chp1, which binds K9-methylated histone H3; Ago1, which binds short interfering (siRNAs); the adaptor protein Tas3, which links Ago1 to Chp1; and centromeric siRNAs. We have generated mutants in RITS to determine the contribution of the two potential chromatin-targeting proteins Chp1 and Ago1 to the centromeric recruitment of RITS. Mutations in Tas3 that disrupt Ago1 binding are permissive for RITS recruitment and maintain centromeric heterochromatin, but the role of Tas3's interaction with Chp1 is unknown. Here, we define the Chp1 interaction domain of Tas3. A strain expressing a tas3 mutant that cannot bind Chp1 (Tas3(Delta)(10-24)) failed to maintain centromeric heterochromatin, with a loss of centromeric siRNAs, a failure to recruit RITS and RDRC to centromeres, and high levels of chromosome loss. These findings suggest a pivotal role for Chp1 and its association with Tas3 for the recruitment of RITS, RDRC, and histone H3 K9 methyltransferase activity to centromeres.
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PMID:Chp1-Tas3 interaction is required to recruit RITS to fission yeast centromeres and for maintenance of centromeric heterochromatin. 1821 52

The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap ((7Me)GpppAC(3-6)) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2'O position of the first transcribed nucleotide, thus converting (7Me)GpppAC(3-6) into (7Me)GpppA(2')(O)(Me)C(3-6). The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2'O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2'O methylation follows an order of events in which 2'O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.
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PMID:Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. 1841 74

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2'-OH methylations of the viral RNA cap (GpppA-RNA-->m(7)GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5'-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5'-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5' stem-loop of the genomic RNA.
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PMID:Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA. 1844 28

RNA interference (RNAi) is a unique gene regulation mechanism in eukaryotic organisms. It is typically mediated by short interfering RNAs (siRNAs), microRNAs (miRNAs), and recently discovered PIWI-interacting RNAs (piRNAs). In plants, RNAi is also called post-transcriptional gene silencing (PTGS), in which diverse small RNAs are utilized to defend against viral or bacterial infection, and to modulate endogenous gene expression and chromatin structure. Many PTGS-related genes have been identified by genetic screens in plants, including suppressor of gene silencing 2 (SGS2, also called RDR6), SGS3, hua enhancer 1 (HEN1), dicer-like 4 (DCL4), and argonaute 1 (AGO1). For most of these proteins, their potential functions have been uncovered through sequence similarity searches and protein domain examinations. For instance, SGS2 encodes a RNA-dependent RNA polymerase (RdRp); HEN1 is a RNA methyltransferase with a double-stranded RNA-binding motif; DCL4 is a Dicer for producing siRNAs; and AGO1 is a RNA slicer with PIWI and PAZ domains. Their retrieved homologs in other organisms including mammals, Drosophila and the nematode Caenorhabditis elegans have also been shown to be crucial in RNAi pathways, indicating an evolutionary conservation for RNAi mechanisms. However, one exception is the SGS3 protein, whose specific function remains unclear so far.
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PMID:The XS domain of a plant specific SGS3 protein adopts a unique RNA recognition motif (RRM) fold. 1863 57

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.
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PMID:Molecular targets for flavivirus drug discovery. 1879 13

ABSTRACT Grapevine rupestris stem pitting (RSP) is a graft-transmissible disease of unknown etiology. We have characterized a virus associated with this disease. The entire genomic sequence (GenBank accession number AF026278) consisted of 8,725 nucleotides excluding a poly(A) tail. Six open reading frames (ORF) were found. ORF1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a RNA-dependent RNA polymerase domain; ORF2, ORF3, and ORF4 compose a triple-gene block; ORF5 encodes a coat protein; and ORF6 is located near the 3' end with unknown function. Sequence analysis indicated that the virus is most similar to apple stem-pitting virus and may be allied with the carla- and potexviruses and grouped with other viruses that infect woody hosts. A specific reverse-transcription polymerase chain reaction (RT-PCR)-based detection method was developed. Among 62 grapevine sources known to be infected with rupestris stem-pitting disease, 60 sources tested positive by RT-PCR. Among 43 healthy vines tested, all were negative. The name grapevine rupestris stem-pitting-associated virus is proposed.
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PMID:Nucleotide sequence and rt-PCR detection of a virus associated with grapevine rupestris stem-pitting disease. 1894 59

Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the "alphavirus-like" supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.
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PMID:A novel mycovirus that is related to the human pathogen hepatitis E virus and rubi-like viruses. 1907 34


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