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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
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PMID:ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture. 1618 75

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'-->5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of approximately 8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.
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PMID:Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. 1654 95

Much progress has been made in understanding the role of structural and accessory proteins in the pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) infections. The SARS epidemic also brought new attention to the proteins translated from ORF1a and ORF1b of the input genome RNA, also known as the replicase/transcriptase gene. Evidence for change within the ORF1ab coding sequence during the SARS epidemic, as well as evidence from studies with other coronaviruses, indicates that it is likely that the ORF1ab proteins play roles in virus pathogenesis distinct from or in addition to functions directly involved in viral replication. Recent reverse genetic studies have confirmed that proteins of ORF1ab may be involved in cellular signaling and modification of cellular gene expression, as well as virulence by mechanisms yet to be determined. Thus, the evolution of the ORF1ab proteins may be determined as much by issues of host range and virulence as they are by specific requirements for intracellular replication.
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PMID:SARS coronavirus replicase proteins in pathogenesis. 1739 59

Masked palm civets are highly susceptible to infection with the severe acute respiratory syndrome coronavirus (SARS-CoV). Infected animals become less aggressive and develop pyrexia, lethargy and diarrhoea. The present study describes the spectrum of histopathological changes in the lung, spleen, lymph node, liver, small intestine, kidney and cerebrum of civets infected experimentally with SARS-CoV. In-situ hybridization (ISH) with probes specific for the RNA polymerase gene demonstrated viral RNA in the lung, small intestine and cerebrum only. In-situ labelling was employed in order to demonstrate cellular apoptosis in the cerebrum, but there was no evidence of apoptosis within the myocardium. These results indicate that SARS-CoV causes multi-organ pathology in civets, similar to that observed in human SARS patients. These parallels suggest that civets may be used as an animal model of this infection to gain insight into the pathogenesis of SARS and for evaluation of candidate vaccines and antiviral drugs.
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PMID:Pathological changes in masked palm civets experimentally infected by severe acute respiratory syndrome (SARS) coronavirus. 1834 98

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

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.
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PMID:SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. 1845 81

Coronaviruses have the potential to cause significant economic, agricultural and health problems. The severe acute respiratory syndrome (SARS) associated coronavirus outbreak in late 2002, early 2003 called attention to the potential damage that coronaviruses could cause in the human population. The ensuing research has enlightened many to the molecular biology of coronaviruses. A programmed -1 ribosomal frameshift is required by coronaviruses for the production of the RNA dependent RNA polymerase which in turn is essential for viral replication. The frameshifting signal encoded in the viral genome has additional features that are not essential for frameshifting. Elucidation of the differences between coronavirus frameshift signals and signals from other viruses may help our understanding of these features. Here we summarize current knowledge and add additional insight regarding the function of the programmed -1 ribosomal frameshift signal in the coronavirus lifecycle.
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PMID:The role of programmed-1 ribosomal frameshifting in coronavirus propagation. 1850 52

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.
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PMID:Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease. 1929

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core)") of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively) revealed that SUD(core) forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core) as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
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PMID:The SARS-unique domain (SUD) of SARS coronavirus contains two macrodomains that bind G-quadruplexes. 1943 9

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.
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PMID:Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo. 1969 90


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