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)

Heterosigma akashiwo (Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family (Marnaviridae), related to picorna-like viruses.
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PMID:Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo. 1501 44

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

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 two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.
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PMID:Comparisons of physical separation methods of Kunjin virus-induced membranes. 1528 61

We report the first discovery and genome sequence of a virus infecting the red imported fire ant, Solenopsis invicta. The 8026 nucleotide, polyadenylated, RNA genome encoded two large open reading frames (ORF1 and ORF2), flanked and separated by 27, 223, and 171 nucleotide untranslated regions, respectively. The predicted amino acid sequence of the 5' proximal ORF1 (nucleotides 28 to 4218) exhibited significant identity and possessed consensus sequences characteristic of the helicase, cysteine protease, and RNA-dependent RNA polymerase sequence motifs from picornaviruses, picorna-like viruses, comoviruses, caliciviruses, and sequiviruses. The predicted amino acid sequence of the 3' proximal ORF2 (nucleotides 4390-7803) showed similarity to structural proteins in picorna-like viruses, especially the acute bee paralysis virus. Electron microscopic examination of negatively stained samples from virus-infected fire ants revealed isometric particles with a diameter of 31 nm, consistent with Picornaviridae. A survey for the fire ant virus from areas around Florida revealed a pattern of fairly widespread distribution. Among 168 nests surveyed, 22.9% were infected. The virus was found to infect all fire ant caste members and developmental stages, including eggs, early (1st-2nd) and late (3rd-4th) instars, worker pupae, workers, sexual pupae, alates ( male symbol and female symbol ), and queens. The virus, tentatively named S. invicta virus (SINV-1), appears to belong to the picorna-like viruses. We did not observe any perceptible symptoms among infected nests in the field. However, in every case where an SINV-1-infected colony was excavated from the field with an inseminated queen and held in the laboratory, all of the brood in these colonies died within 3 months.
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PMID:A picorna-like virus from the red imported fire ant, Solenopsis invicta: initial discovery, genome sequence, and characterization. 1538 Mar 66

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.
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PMID:Multiple virus infections in the honey bee and genome divergence of honey bee viruses. 1557 17

In this study, Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis omega virus (NomegaV). DpTV particles are isometric, with a diameter of about 40 nm and a buoyant density of 1.281 g cm(-3) in CsCl. The virus has two capsid proteins (of 62 500 and 6800 Da) and two single-stranded RNA molecules (RNA1 and RNA2), which are 5492 and 2490 nt long, respectively. RNA1 has a large open reading frame (ORF) encoding a polypeptide of 180 kDa; RNA2 contains two partially overlapping ORFs encoding polypeptides of 17 and 70 kDa. The 180 kDa protein, which contains consensus motifs of a putative methyltransferase, helicase and RNA-dependent RNA polymerase, shows significant similarity to those of other tetraviruses. The 17 kDa protein is a PEST (Pro/Glu/Ser/Thr) protein of unknown function. The 70 kDa protein is the coat protein precursor and is predicted to be cleaved at an Asn-Phe site located after residue 570. The 70 kDa protein shows 86 and 66 % identity to its homologues in NomegaV and Helicoverpa armigera stunt virus, respectively. Secondary-structure analysis revealed that the RNAs of DpTV have tRNA-like structures at their 3' termini.
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PMID:Isolation and identification of a new tetravirus from Dendrolimus punctatus larvae collected from Yunnan Province, China. 1572 41

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

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.
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PMID:Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase. 1599 62

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).
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PMID:Complete nucleotide sequence and molecular probing of potato virus S genome. 1617 17


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