Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.
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PMID:Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein. 1291 29

The identification and development of new antiviral agents that can be used to combat hepatitis C virus (HCV) infection has been complicated by both technical and logistic issues. There are few, if any, robust methods by which HCV virions can be grown in vitro. The development of HCV RNA replicons has been a great breakthrough that has allowed for the undertaking of significant screening efforts to identify inhibitors of HCV intracellular replication. However, since replicons do not undergo a complete replication cycle, drug screening programs and mechanism of action studies based solely on these assays will not identify compounds targeting either early (virion attachment, entry, uncoating) or late (virion assembly, egress) stages of the viral replication cycle. Drugs that negatively affect the infectivity of new virions will also not be identified using HCV RNA replicons. Bovine viral diarrhea virus (BVDV) shares a similar structural organization with HCV, and both viruses generally cause chronic long-term infections in their respective hosts. The BVDV surrogate model is attractive, since it is a virus-based system. It is easy to culture the virus in vitro, molecular clones are available for genetic studies, and the virus undergoes a complete replication cycle. Like HCV, BVDV utilizes the LDL receptor to enter cells, uses a functionally similar internal ribosome entry site (IRES) for translation, uses an NS4A cofactor with its homologous NS3 protease, has a similar NS3 helicase/NTPase, a mechanistically similar NS5B RNA-dependent RNA polymerase, and a seemingly equivalent mechanism of virion maturation, assembly and egress. While the concordance between drugs active in either BVDV or HCV is largely unknown at this time, BVDV remains a popular model system with which drugs can be evaluated for potential antiviral activity against HCV and in studies of drug mechanism of action.
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PMID:Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents. 1451 16

Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.
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PMID:Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein. 1455 63

Rotaviruses, members of family Reoviridae, are a major cause of acute gastroenteritis of infants and young children. The rotavirus genome consists of 11 segments of double-stranded (ds)RNA and the virion is an icosahedron composed of multiple layers of protein. The virion core is formed by a layer of VP2 and contains multiple copies of the RNA-dependent RNA polymerase VP1 and the mRNA-capping enzyme VP3. Double-layered particles (DLPs), representing cores surrounded by a layer of VP6, direct the synthesis of viral mRNAs. Rotavirus core- and DLP-like replication intermediates (RIs) catalyze the synthesis of dsRNA from viral template mRNAs coincidentally with the packaging of the mRNAs into the pre-capsid structures of RIs. In addition to structural proteins, the nonstructural proteins NSP2 and NSP5 are components of RIs with replicase activity. NSP2 self assembles into octameric structures that have affinity for ssRNA and NTPase and helix-destabilizing activites. Its interaction with nucleotides induces a conformational shift in the octamer to a more condensed form. Phosphate residues generated by the NTPase activity are believed to be transferred from NSP2 to NSP5, leading to the hyperphosphorylation of the latter protein. It is suspected that the transfer of the phosphate group to NSP5 allows NSP2 to return to its noncondensed state and, thus, to accept another NTP molecule. The NSP5-mediated cycling of NSP2 from condensed to noncondensed combined with its RNA binding and helix-destabilizing activities are consistent with NSP2 functioning as a molecular motor to facilitate the packaging of template mRNAs into the pre-capsid structures of RIs. Similarities with the bluetongue virus protein NS2 and the reovirus proteins sigmaNS and micro2 suggest that they may be functional homologs of rotavirus NSP2 and NSP5.
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PMID:Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae. 1501 Feb 17

Double-stranded RNA viruses infecting bacterial hosts belong to the Cystoviridae family. Bacteriophage phi6 is one of the best characterized dsRNA viruses and shares structural as well as functional similarities with other well-studied eukaryotic dsRNA viruses (e.g. L-A, rotavirus, bluetongue virus, and reovirus). The assembly pathway of the enveloped, triple-layered phi6 virion has been well documented and can be divided into four distinct steps which are (1) procapsid formation, (2) genome encapsidation and replication, (3) nucleocapsid surface shell assembly, and (4) envelope formation. In this review, we focus primarily on the procapsid and nucleocapsid assembly for which in vitro systems have been established. The in vitro assembly systems have been instrumental in revealing assembly intermediates and conformational changes that are common to phi6 and phi8, two cystoviruses with negligible sequence homology. Two viral enzymes, the packaging NTPase (P4) and the RNA-dependent RNA polymerase (P2), were found essential for the nucleation step. The nucleation complex contains one or more tetramers of the major procapsid protein (P1) and is further stabilized by protein P4. Interaction of P1 and P4 during assembly is accompanied by an additional folding of their respective polypeptide chains. The in vitro assembled procapsids were shown to selectively package and replicate the genomic ssRNA. Furthermore, in vitro assembly of infectious nucleocapsids has been achieved in the case of phi6. The in vitro studies indicate that the nucleocapsid coat protein (P8) assembles around the polymerase complex in a template-assisted manner. Implications for the assembly of other dsRNA viruses are also presented.
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PMID:Self-assembly of double-stranded RNA bacteriophages. 1501 Feb 20

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
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PMID:Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. 1514 Sep 59

Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, inhibits reovirus replication and viral RNA and protein production. In mouse L929 cells, antiviral effects were greatest at 30 microg of MPA/ml. At this dosage, MPA inhibited replication of reovirus strain T3D more than 1,000-fold and inhibited replication of reovirus strain T1L nearly 100-fold, compared to non-drug-treated controls. Genetic reassortant analysis indicated the primary determinant of strain-specific differences in sensitivity to MPA mapped to the viral M1 genome segment, which encodes the minor core protein mu2. MPA also inhibited replication of both strains of reovirus in a variety of other cell lines, including Vero monkey kidney and U373 human astrocytoma cells. Addition of exogenous guanosine to MPA-treated reovirus-infected cells restored viral replicative capacity to nearly normal levels. These results suggest the mu2 protein is involved in the uptake and processing of GTP in viral transcription in infected cells and strengthens the evidence that the mu2 protein can function as an NTPase and is likely a transcriptase cofactor.
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PMID:Inhibition of reovirus by mycophenolic acid is associated with the M1 genome segment. 1516 10

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.
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PMID:Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins. 1525 67

The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in (1171)Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH(2)-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.
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PMID:Proteolytic processing of sapovirus ORF1 polyprotein. 1591 82

Positive-sense single-stranded RNA (+ssRNA) viruses replicate by uncoating the RNA genome for translation to provide viral proteins essential for genome replication and the production of new viral particles. The viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently. The synthesized proteins include viral RNA-dependent RNA polymerase (RdRP), viral genome-linked protein (VPg), and a helicase. VPg is covalently attached to the genomic form of +ssRNA viruses. Helicases and NTPase unwind the RNA before replication. VPg and helicases have been identified in +ssRNA families, however, the presence of VPg and helicase in the Astroviridae, another +ssRNA family, has not been fully elucidated. Computational tools were utilized to provide sequence analysis evidence for the presence and genomic location of astrovirus VPg and helicase. HMMER program v2.1.1 was used to build Hidden Markov Model (HMM) profile for calicivirus VPg to search for conserved motifs in the astrovirus genome. We performed phylogenetic analysis of two genomic regions of astroviruses and caliciviruses (encoding the RdRP and VPg). We identified a putative VPg coding region in astrovirus. This region was located in open reading frame 1a (ORF1 a) and included sites with high sequence similarity to the VPg coding regions of Caliciviridae, Piconaviridae, and Potyviridae. A region encoding a putative astrovirus helicase identified conserved motifs only with pestivirus helicase sequences. Sequence analysis and comparison to other +ssRNA viruses supports the presence of VPg in the Astroviridae. Further laboratory analysis will be necessary to confirm these findings.
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PMID:Genome prediction of putative genome-linked viral protein (VPg) of astroviruses. 1596 5


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