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)

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.
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PMID:Nuclear localization of flavivirus RNA synthesis in infected cells. 1669 25

The global prevalence of hepatitis C virus (HCV) infection and serious health consequences associated with chronic state of the disease have become a significant health problem worldwide. Currently, there is no vaccine to prevent the disease and no specific antiviral drug directed against HCV infection. The current standard of care, interferon-based therapies, both alone or in combination with ribavirin, has demonstrated limited success and is associated with undesirable side effects. Thus, the treatment of the chronic HCV infection represents an unmet medical need. With advances in the understanding of HCV replication and the crystal structures of the virally encoded enzymes, the HCV NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase have emerged as ideal targets toward the control of the disease and the development of new anti-HCV agents. In this review, we will summarize the current treatment options, and outline the approaches toward discovery of small molecule antivirals against the virally encoded enzymes. The current clinical studies of promising lead compounds are also reviewed.
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PMID:Recent development of therapeutics for chronic HCV infection. 1682 88

Recently, nitric oxide (NO) has been shown to suppress dengue virus (DENV) RNA and protein accumulation in infected cells. In this report, the potential target of the inhibitory effect of NO was studied at the molecular level. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), showed an inhibitory effect on RNA accumulation at around 8-14 h post-infection, which corresponded to the step of viral RNA synthesis in the DENV life cycle. The activity of the viral replicase isolated from SNAP-treated DENV-2-infected cells was suppressed significantly compared with that of the negative-control N-acetyl-DL-penicillamine (NAP)-treated cells. Further investigations on the molecular target of NO action showed that the activity of recombinant DENV-2 NS5 in negative-strand RNA synthesis was affected in the presence of 5 mM SNAP in in vitro RNA-dependent RNA polymerase (RdRp) assays, whereas the RNA helicase activity of DENV-2 NS3 was not inhibited up to a concentration of 15 mM SNAP. These results suggest that the inhibitory effect of NO on DENV infection is partly via inhibition of the RdRp activity, which then downregulates viral RNA synthesis.
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PMID:Antiviral action of nitric oxide on dengue virus type 2 replication. 1696 59

Flaviviruses are enveloped viruses with a single-stranded, 10.7kb positive-sense RNA genome. The genomic RNA, which has a 5' cap but no poly(A) tail, is translated as a single polyprotein that is then cleaved into three structural proteins and seven non-structural (NS) proteins by both viral and host proteases. The NS proteins include an RNA-dependent RNA polymerase (NS5), a helicase/protease (NS3), and other proteins that form part of the viral replication complex. Sequences and structures in the 5' and 3' untranslated regions (UTR) and capsid gene, including the cyclization sequences, the upstream AUG region, and the terminal 3' stem-loop, regulate translation, RNA synthesis and viral replication. We have also found that an RNA hairpin structure in the capsid coding region (cHP) influences start codon selection and viral replication of the flavivirus dengue virus (DENV). Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) were used to further dissect the role of conserved regions of the 5' and 3' UTRs; several P-PMOs were shown to specifically inhibit DENV translation and/or RNA synthesis and, hence, are potentially useful as antiviral agents. Regarding the mechanism of DENV translation, we have shown that DENV undergoes canonical cap-dependent translation initiation as well as a non-canonical mechanism when cap-dependent translation is suppressed. Although much remains to be elucidated about the molecular biology of flavivirus infection, progress is being made towards defining the cis and trans factors that regulate flavivirus translation and replication.
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PMID:Molecular biology of flaviviruses. 1731 52

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin. Triton X-100 (TX100) treatment reduced RdRp activity by half in WNV but totally destroyed RdRp activity in JEV. TX100 also dissociated NS1' in addition to NS1 from NS5 and NS3 inJEV. Antibodies to NS3 coprecipitated NS1' along with NS5 only from DOC-solubilized but not from TX100-treated extracts, the former of which alone retained RdRp activity. Exogenous addition of recombinant NS1' to TX100 treated JEV-induced membranes restored the defect in the release step of RNA synthesis. Our results suggest for the first time a direct role for JEV NS1' in viral RNA synthesis in vitro.
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PMID:Organization of flaviviral replicase proteins in virus-induced membranes: a role for NS1' in Japanese encephalitis virus RNA synthesis. 1731 59

Since the discovery of the hepatitis C virus (HCV) as the major cause of non-A, non-B hepatitis in 1989, the search for specific targeted antiviral therapy for HCV (STAT-C) has been underway. Recently, major advances in the understanding of HCV biology and the development of an in vitro system of HCV replication have contributed to the selection of multiple candidate drugs for the treatment of hepatitis C. In 2006, five such candidate drugs have entered phase II clinical trials in patients chronically infected with hepatitis C, including small molecule inhibitors of the HCV NS3 serine protease and NS5B RNA-dependent RNA polymerase. This review focuses on hepatitis C protease and polymerase inhibitors that have progressed to phase II clinical development, foreshadowing the era of STAT-Cs.
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PMID:Specific targeted antiviral therapy for hepatitis C. 1733 72

Four novel series of base modified ribonucleoside analogues were synthesized and evaluated as potential anti-HCV agents. For two compounds notable anti-HCV activity was observed The triphosphates of bicyclic pyrimidine ribonucleosides were studied as substrates/inhibitors of HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) and RNA helicase/NTPase (NS3 protein).
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PMID:Base-modified ribonucleosides as potential anti-hepatitis C virus agents. 1877 32

Flaviviruses are a major cause of infectious disease in humans. Dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. West Nile virus, which recently spread from the Mediterranean basin to the Western Hemisphere, now causes thousands of sporadic cases of encephalitis annually. Despite the existence of licensed vaccines, yellow fever, Japanese encephalitis and tick-borne encephalitis also claim many thousands of victims each year across their vast endemic areas. Antiviral therapy could potentially reduce morbidity and mortality from flavivirus infections, but no effective drugs are currently available. This article introduces a collection of papers in Antiviral Research on molecular targets for flavivirus antiviral drug design and murine models of dengue virus disease that aims to encourage drug development efforts. After reviewing the flavivirus replication cycle, we discuss the envelope glycoprotein, NS3 protease, NS3 helicase, NS5 methyltransferase and NS5 RNA-dependent RNA polymerase as potential drug targets, with special attention being given to the viral protease. The other viral proteins are the subject of individual articles in the journal. Together, these papers highlight current status of drug discovery efforts for flavivirus diseases and suggest promising areas for further research.
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PMID:Molecular targets for flavivirus drug discovery. 1879 13

HCV is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans. Although all HCV enzymes are, in theory, equally appropriate for therapeutic intervention, the NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase are the most popular targets from a drug-discovery perspective. A number of active-site inhibitors of the NS3 protease as well as allosteric inhibitors of the NS5B polymerase are being developed. We determined the crystal structures of complexes of NS3/NS4A/active-site inhibitor as well as NS5B/allosteric inhibitor to permit structure-based drug design and the efficient optimization of leads. The methods for obtaining such structures by crystal soaking procedures are described.
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PMID:Preparation and handling of hepatitis C viral proteins NS3 and NS5B for structural studies. 1900 57


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