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)

The family of the Flaviviridae contains 3 genera: (i) the hepaciviruses, to which belongs Hepatitis C virus (HCV), (ii) the flaviviruses and (iii) the pestiviruses. Over 140 million people, more than four times the number of HIV-positive individuals, are chronically infected with the HCV. Hepatitis G virus (HGV) has not yet been assigned to a genus. The impact of this recently discovered virus is yet to be established. Infections with flaviviruses such as Yellow Fever virus (YFV), Dengue Fever virus (DENV), Japanese Encephalitis virus (JEV) and Tick-borne Encephalitis virus (TBEV) are emerging world-wide. The Pestiviruses, Bovine Viral Diarrhea virus (BVDV), Classical Swine Fever virus (CSFV) and Border Disease virus (BDV) have a serious impact on life-stock. At present, only treatment with interferon, alone or combined with ribavirin, has been approved for the treatment of HCV infections. No specific antivirals are available for the treatment of infections with Hepaci-, Flavi- or Pestiviruses. Possible targets for inhibition of the replication of Flaviviridae are the binding to, and the uptake of the virus in the cell; the internal ribosomal entry site (IRES) of Hepaci- and Pestiviruses; viral proteases; the viral RNA-dependent RNA polymerase and the viral helicase. The search for specific inhibitors of HCV replication is hindered by the absence of an efficient cell culture system for propagation of this virus. In addition, small laboratory animals, including mice, are not susceptible to HCV infection. Flaviviruses may cause infection in mice, but do so mainly following direct intracerebral inoculation. We have established a small animal model for flavivirus infections in SCID mice inoculated peripherally with the murine flavivirus Modoc.
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PMID:Infections with flaviviridae. 1065 76

The NS5B gene, cloned from Classical swine fever virus (CSFV) genome, was expressed in porcine kidney cells PK-15, natural host of CSFV. In purifying cytoplasmic extracts from these cells by means of different concentrations of salt, glycerol and detergent four fractions, namely crude supernatant (SC) and different purified supernatants (S1, S2 and S3) were obtained. Using Western blot analysis the NS5B protein was found in all these fractions, showing that it was soluble in both higher and lower concentrations of salt, glycerol and detergent. The NS5B protein present in the four different fractions exhibited RNA-dependent RNA polymerase (RdRp) activity, but it was unable to complete the whole process of RNA synthesis. Site-directed mutation analysis showed that Thy216 and Cyt228 were essential for RNA synthesis while Cyt219 was not, suggesting that CSFV RdRp was template-specific. We conclude that initiation of RNA synthesis by CSFV RdRp includes also template priming.
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PMID:RNA-dependent RNA polymerase activity of Classical swine fever virus NS5B protein expressed in natural host cells. 1452 73

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5Bdelta24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus- or minus-strand viral RNA synthesis de novo in a primer-independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA-dependent RNA polymerase in vitro. Both Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm GTP, and a 3'-terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North-Western blot analysis, to specifically interact with the 3'-end of both plus- and minus-strand viral RNA templates.
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PMID:De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase. 1465 21

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.
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PMID:De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase. 1602 97

To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of Classical swine fever virus was performed. Results show that the N-terminal domain (positions 1-300) of the protein might be divided artificially into four different regions, N1-N4. The N1 region (positions 1-61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for de novo RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63-216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217-280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281-300) reduced elongative synthesis and destroyed de novo RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and de novo RNA synthesis.
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PMID:Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymerase. 1643 21

The RNA-dependent RNA polymerase (RdRp) in viruses of the family Flaviviridae plays an important role in the viral replication process and in the forming of a replicase complex. We used small interfering RNAs (siRNAs) corresponding to the highly conservative Motif V of RdRp gene of different viruses to examine their role in modulating the expression of RdRp. Evaluation of the expression of RdRps was performed by the fluorescence, flow cytometry, Western blotting, and real-time PCR. We found that Classical swine fever virus (CSFV) siRNA could completely block the transcription and expression of RdRp. Additionally, Hepatitis C virus (HCV) siRNA could cause effective inhibition of RdRp, whereas Japanese encephalitis virus siRNA did not show significant repression of corresponding RdRp. These results demonstrated that siRNAs inhibited the expression of tested RdRps at the transcription level or at the posttranscriptional processing to a different extent.
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PMID:Inhibition of expression of rna polymerase with small interfering RNAs targeting a conserved motif in the respective viral genes in viruses of the family Flaviviridae. 1807 10

Classical swine fever virus (CSFV) nonstructural protein 3 (NS3) is believed to possess three enzyme activities that are likely to be essential for virus replication: a serine protease located in the N-terminus and NTPase as well as helicase activities located in the C-terminus. In this report, we expressed NS3 helicase domain (NS3h) in E. coli and characterized its helicase activity. The NS3h helicase activity was dependent on the presence of NTP and divalent cations, with a preference for ATP and Mn(2+), and required the substrates possessing a 3' un-base-paired region on the RNA template strand. The NS3h helicase activity was proportional to increasing lengths of the 3' un-base-paired regions up to 16 nucleotides of the RNA substrates. We also investigated the modulation of NS3 NTPase/helicase activities by NS3 protease domain and NS5B, an RNA-dependent RNA polymerase (RdRp). Our data showed that the NS3 protease domain enhanced the helicase activity of NS3 but had no effect on its NTPase activity. For the truncated NS3 (helicase domain, NS3h), both NTPase and helicase activities were up-regulated by NS5B. However, for the full-length NS3 (NS3FL), the NTPase activity, but not the helicase activity, was stimulated by NS5B. Maltose-binding protein (MBP) pull-down as well as enzyme-linked immunosorbent assays confirmed the specific interaction between NS3 and NS5B.
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PMID:Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase. 1918 95

Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.
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PMID:Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B. 2648 49

Classical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by a de novo mechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCE Pigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genus Pestivirus, family Flaviviridae Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.
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PMID:Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain. 2972 May 18