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

---

Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.
...
PMID:Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. 1050 28

Hepatitis C virus (HCV) has a positive-stranded RNA genome of about 9.5 kb and a large open reading frame encoding a precursor polyprotein of ca. 3,000 amino acids (aa). This polyprotein is cleaved by host cellular signalase(s) and viral proteases into 10 viral proteins in the order of NH(2)-Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS 5B-COOH. Core and E1/E2 are considered to be a capsid protein and envelope glycoproteins, respectively. NS2-NS5B are putative nonstructural proteins involved in the replication of HCV. NS2/3 is a metalloprotease which cleaves in cis at the NS2/3 junction. NS3 possesses serine protease and RNA helicase activities and is responsible for the cleavage of the remaining nonstructural proteins. NS4A is suggested to be a cofactor for NS3 protease. Although the function of p7, NS4B and NS5A are still unknown, an association of a mutation in NS5A with a susceptibility to interferon (IFN) has been reported. NS5B possesses an RNA-dependent RNA polymerase activity. Most of the current findings in HCV proteins depend on expression studies of HCV cDNA clones because of the lack of an efficient replication system in cell cultures. Therefore, a final assignment of cleavages and functions of HCV proteins has to await the propagation of HCV in cell cultures.
...
PMID:Processing and functions of Hepatitis C virus proteins. 1051 68

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.
...
PMID:Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. 1054 80

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.
...
PMID:Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. 1055 68

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.
...
PMID:Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. 1055 28

Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Therapeutic options for hepatitis C are limited. Standard monotherapy with interferon-alpha leads to a sustained response in only 10-20% of patients. Recent studies have shown improved sustained response rates for the combination of interferon-alpha and ribavirin. Despite these improvements, more effective therapies are needed. A variety of alternative agents are currently being evaluated in clinical trials. Recent advances in the molecular virology of hepatitis C have identified specific antiviral targets such as the viral NS3 serine protease, the RNA helicase, and the RNA-dependent RNA polymerase. In addition, gene therapeutic strategies aimed at inhibiting HCV gene expression and replication as well as immunotherapeutic concepts aimed at enhancing the cellular immune response against HCV are being explored in various experimental systems. These and other novel antiviral strategies may complement the existing therapeutic modalities in the future.
...
PMID:Current and evolving therapies for hepatitis C. 1056 26

The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae. By using heterologously expressed proteins, we demonstrate that the 77 kDa NS5B protein of two pestiviruses, bovine viral diarrhoea virus and classical swine fever virus, and the 100 kDa NS5 protein of the West Nile flavivirus possess RdRp activity in vitro. As originally shown for the RdRp of hepatitis C virus, RNA synthesis catalysed by the pestivirus and flavivirus enzymes is strictly primer-dependent in vitro. Accordingly, initiation of RNA polymerization on homopolymeric RNAs and heteropolymeric templates, the latter with a blocked 3'-hydroxyl group, was found to be dependent on the presence of complementary oligonucleotide primer molecules. On unblocked heteropolymeric templates, including authentic viral RNAs, the RdRps were shown to initiate RNA synthesis via intramolecular priming at the 3'-hydroxyl group of the template and 'copy-back' transcription, thus yielding RNase-resistant hairpin molecules. Taken together, the RdRps of different members of the Flaviviridae were demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid-polymerizing enzymes.
...
PMID:The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro. 1057 50

Infection with the hepatitis C virus (HCV) is the major cause of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in vitro, several of its key features have been elucidated in the past few years. The viral genome is a positive-sense, single-stranded, 9.6 kb long RNA molecule. The viral genome is translated into a single polyprotein of about 3000 amino acids. The viral polyprotein is proteolytically processed by the combination of cellular and viral proteinases in order to yield all the mature viral gene products. The genomic order of HCV has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C, E1 and E2 are the virion.structural proteins. The function of p7 is currently unknown. These proteins have been shown to arise from the viral polyprotein via proteolytic processing by the host signal peptidases. Generation of the mature nonstructural proteins, NS2 to NS5B, relies on the activity of viral proteinases. Cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine proteinase also contained within the N-terminal region of NS3. NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the proteinase. While no function has yet been attributed to NS4B, it has recently been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase.
...
PMID:Molecular virology of the hepatitis C virus. 1062 60

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.
...
PMID:De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. 1062 48

The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Worldwide, more than 170 million people are chronically infected with Hepatitis C virus and are at risk of developing cirrhosis and/or liver cancer. In addition, infections with arthropod-borne flaviviruses (such as dengue fever, Japanese encephalitis, tick-borne encephalitis, St. Louis encephalitis, Murray Valley encephalitis, West Nile, and yellow fever viruses) are emerging throughout the world. The pestiviruses have a serious impact on livestock. Unfortunately, no specific antiviral therapy is available for the treatment or the prevention of infections with members of the Flaviviridae. Ongoing research has identified possible targets for inhibition, including binding of the virus to the cell, uptake of the virus into the cell, the internal ribosome entry site of hepaciviruses and pestiviruses, the capping mechanism of flaviviruses, the viral proteases, the viral RNA-dependent RNA polymerase, and the viral helicase. In light of recent developments, the prevalence of infections caused by these viruses, the disease spectrum, and the impact of infections, different strategies that could be pursued to specifically inhibit viral targets and animal models that are available to study the pathogenesis and antiviral strategies are reviewed.
...
PMID:Perspectives for the treatment of infections with Flaviviridae. 1062 92


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---