Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here a novel approach for detecting and quantitating human respiratory syncytial virus (RSV) based on expression of a reporter gene from an RSV minigenome. BHK cells were cytoplasmically transformed with a noncytopathic Sindbis virus replicon expressing T7 RNA polymerase. These cells were then cotransfected with T7 expression plasmids that contain the cDNA of an RSV minigenome and the genes for RSV nucleocapsid proteins N, P, and L. The minigenome contains a reporter gene such as lacZ or CAT flanked by cis-acting RSV transcription signals. Subsequent infection of these cells with RSV resulted in a high level of reporter gene expression which could be inhibited by ribavirin. Mock-infected cells exhibited background levels of expression. This assay can be used to quantitate RSV and titer neutralizing antibody and may be a valuable tool for screening compounds for anti-RSV activity. It serves as a prototype for other negative-strand RNA viruses.
 ...
PMID:Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses. 981 15

We performed a large-scale random screening of an in-house chemical library based on the inhibition of respiratory syncytial virus (RSV)-induced cytopathic effect on HeLa (human cervical carcinoma) cells, and found a novel and specific anti-RSV agent, 6-{4-[(biphenyl-2-ylcarbonyl) amino]benzoyl}-N-cyclopropyl-5,6-dihydro-4H-thieno[3,2-d][1]benzazepine-2-carboxamide (YM-53403). YM-53403 potently inhibited the replication of RSV strains belonging to both A and B subgroups, but not influenza A virus, measles virus, or herpes simplex virus type 1. A plaque reduction assay was used to determine the 50% effective concentration (EC(50)) value for YM-53403. The value, 0.20 microM, was about 100-fold more potent than ribavirin. The result of a time-dependent drug addition test showed that YM-53403 inhibited the life cycle of RSV at around 8h post-infection, suggesting an inhibitory effect on early transcription and/or replication of the RSV genome. Consistent with this result, two YM-53403-resistant viruses have a single point mutation (Y1631H) in the L protein which is a RNA polymerase for both the transcription and replication of the RSV genome. YM-53403 is an attractive compound for the treatment of RSV infection because of its highly potent anti-RSV activity and the new mode of action, which differs from that of currently reported antiviral agents.
 ...
PMID:YM-53403, a unique anti-respiratory syncytial virus agent with a novel mechanism of action. 1570 39

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.
 ...
PMID:Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase. 1618 12

Respiratory syncytial virus (RSV) consists of fusion (F), glyco (G), and small hydrophobic (SH) proteins as envelope proteins, and infects through cell fusion. F protein is expressed on the surface of infected cells, and induces cell fusion. In the present report, expression plasmids of the F, G and SH proteins were constructed and cell fusion activity was investigated under T7 RNA polymerase. F protein alone induced cell fusion at a lower concentration than previously reported, and co-expression of F and SH proteins induced cell fusion more efficiently than F protein alone. Palivizumab is the only prophylactic agent against RSV infection. Palivizumab-resistant strains having mutations of the F protein of K272E and S275F were reported. These mutations were introduced into an F-expression plasmid, and exhibited no inhibition of cell fusion with palivizumab. Among the RSV F protein mutants, N276S has been reported to have partial resistance against palivizumab, but the F expression plasmid with the N276S mutation showed a reduction in cell fusion in the presence of palivizumab, showing no resistance to palivizumab. The present expression system was useful for investigating the mechanisms of RSV cell fusion.
...
PMID:Cell fusion assay by expression of respiratory syncytial virus (RSV) fusion protein to analyze the mutation of palivizumab-resistant strains. 2679 81