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)

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.
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PMID:A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies. 1583 55

The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.
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PMID:Expression, purification, and characterization of SARS coronavirus RNA polymerase. 1584 May 16

When unexpected diseases such as the severe acute respiratory syndrome (SARS) and avian influenza become a serious threat to public health, an immediate response is imperative. This should take into consideration existing licensed antiviral drugs against other viral diseases already known to be safe for use in humans. In this report, evidence is presented that HIV-1 protease inhibitors (PIs) currently used in anti-HIV-1 therapies might exert some effects on SARS and perhaps, on avian influenza. Evidence for the potential benefits of PIs against the SARS coronavirus (SARS-CoV) is provided by empirical clinical studies, in vivo viral inhibition assays and computational simulations of the docking of these compounds to the active site of the main SARS-CoV protease. As suggested by in silico docking of these molecules to a theoretical model of a subunit of type A influenza virus RNA-dependent RNA polymerase, there also exists a remote possibility that these PIs may have an effect on avian influenza viruses. Although this evidence is still far from being definitive, the results so far obtained suggest that PIs should be seriously taken into consideration for further testing as potential therapeutic agents for SARS and avian influenza.
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PMID:Expanding the frontiers of existing antiviral drugs: possible effects of HIV-1 protease inhibitors against SARS and avian influenza. 1589 56

We recently published experimental results that indicated Aurintricarboxylic Acid (ATA) could selectively inhibit SARS-CoV replication inside host cells by greater than 1000 times. This inhibition suggested that ATA could be developed as potent anti-viral drug. Here, to extend our experimental observation, we have incorporated protein structural studies (with positive/negative controls) to investigate the potential binding modes/sites of ATA onto RNA-dependent RNA polymerase (RdRp) from SARS-CoV and other pathogenic positive-strand RNA-viruses, as well as other proteins in SARS-CoV based on the fact that ATA binds to Ca2+-activated neutral protease (m-calpain), the protein tyrosine phosphatase (PTP) and HIV integrase which have existing crystal structures. Eight regions with homologous 3D-conformation were derived for 10 proteins of interest. One of the region, Rbinding (754-766 in SARS-CoV's RdRp), located in the palm sub-domain mainly constituted of anti-parallel beta-strand-turn-beta-strand hairpin structures that covers two of the three RdRp catalytic sites (Asp 760, Asp761), was also predicted by molecular docking method (based on free energy of binding DeltaG) to be important binding motif recognized by ATA. The existence of this strictly conserved region that incorporated catalytic residues, coupled with the homologous ATA binding pockets and their consistent DeltaG values, suggested strongly ATA may be involved in an analogous inhibition mechanism of SARS-COV's RdRp in concomitant to the case in m-calpain, PTP and HIV integrase.
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PMID:Structural analysis of inhibition mechanisms of aurintricarboxylic acid on SARS-CoV polymerase and other proteins. 1597 41

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

Coronavirus replication and transcription machinery involves multiple virus-encoded nonstructural proteins (nsp). We report the crystal structure of the hexadecameric nsp7-nsp8 supercomplex from the severe acute respiratory syndrome coronavirus at 2.4-angstroms resolution. nsp8 has a novel 'golf-club' fold with two conformations. The supercomplex is a unique hollow, cylinder-like structure assembled from eight copies of nsp8 and held tightly together by eight copies of nsp7. With an internal diameter of approximately 30 angstroms, the central channel has dimensions and positive electrostatic properties favorable for nucleic acid binding, implying that its role is to confer processivity on RNA-dependent RNA polymerase.
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PMID:Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer. 1622 2

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

There are a number of antivirals as well as antiviral strategies that could be envisaged to prevent or treat severe acute respiratory syndrome (SARS) (or similar) coronavirus (CoV) infections. Targets for the prophylactic or therapeutic interventions include interaction of the spike (S) glycoprotein (S1 domain) with the host cell receptor, fusion of the S2 domain with the host cell membrane, processing of the replicase polyproteins by the virus-encoded proteases (3C-like cysteine protease [3CLpro] and papain-like cysteine protease) and other virus-encoded enzymes such as the NTPase/helicase and RNA-dependent RNA polymerase. Human monoclonal antibody blocking S1 may play an important role in the immunoprophylaxis of SARS. Fusion inhibitors reminiscent of enfuvirtide in the case of HIV may also be developed for SARS-CoV. Various peptidomimetic and nonpeptidic inhibitors of 3CLpro have been described, the best ones inhibiting SARS-CoV replication with a selectivity index greater than 1000. Human interferons, in particular alpha- and beta-interferon, as well as short interfering RNAs could further be pursued for the control of SARS. Various other compounds, often with an ill-defined mode of action but selectivity indexes up to 100, have been reported to exhibit in vitro activity against SARS-CoV: valinomycin, glycopeptide antibiotics, plant lectins, hesperetin, glycyrrhizin, aurintricarboxylic acid, chloroquine, niclosamide, nelfinavir and calpain inhibitors.
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PMID:Potential antivirals and antiviral strategies against SARS coronavirus infections. 1659 9

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.
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PMID:Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes. 1675 1

Coronaviruses can infect a variety of animals including poultry, livestock, and humans and are currently classified into three groups. The interspecies transmissions of coronaviruses between different hosts form a complex ecosystem of which little is known. The outbreak of severe acute respiratory syndrome (SARS) and the recent identification of new coronaviruses have highlighted the necessity for further investigation of coronavirus ecology, in particular the role of bats and other wild animals. In this study, we sampled bat populations in 15 provinces of China and reveal that approximately 6.5% of the bats, from diverse species distributed throughout the region, harbor coronaviruses. Full genomes of four coronavirues from bats were sequenced and analyzed. Phylogenetic analyses of the spike, envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, revealed that bat coronaviruses cluster in three different groups: group 1, another group that includes all SARS and SARS-like coronaviruses (putative group 4), and an independent bat coronavirus group (putative group 5). Further genetic analyses showed that different species of bats maintain coronaviruses from different groups and that a single bat species from different geographic locations supports similar coronaviruses. Thus, the findings of this study suggest that bats may play an integral role in the ecology and evolution of coronaviruses.
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PMID:Prevalence and genetic diversity of coronaviruses in bats from China. 1684 Mar 28


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