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

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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)

All pathogenic flaviviruses examined thus far inhibit host interferon (IFN) responses by suppressing the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Both Langat virus (LGTV; a member of the tick-borne encephalitis virus serogroup) and Japanese encephalitis virus use the nonstructural protein NS5 to suppress JAK-STAT signaling. However, NS5 is also critical to virus replication, contributing methyltransferase and RNA-dependent RNA polymerase (RdRP) activities. The specific amino acid residues of NS5 involved in IFN antagonism are not known. Here, we demonstrate that the LGTV NS5 JAK-STAT inhibitory domain is contained between amino acids 355 and 735 (of 903), a range which lies within the RdRP domain. Furthermore, we identified two noncontiguous stretches of specific amino acids within the RdRP, 374 to 380 and 624 to 647, as critical for inhibition of JAK-STAT signaling. Despite considerable separation on the linear NS5 sequence, these residues localized adjacent to each other when modeled on the West Nile virus RdRP crystal structure. Due to the general conservation of RdRP structures, these results suggest that the specific residues identified act cooperatively to form a unique functional site on the RdRP responsible for JAK-STAT inhibition. This insight into the mechanism underlying flavivirus IFN evasion strategies will facilitate the design of antiviral therapeutics that potentiate the action of IFN during infection.
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PMID:Identification of residues critical for the interferon antagonist function of Langat virus NS5 reveals a role for the RNA-dependent RNA polymerase domain. 1745 29

The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn(2+) was found to be 20 times more effective than Mg(2+) in coordinating the catalytic reaction of RdRp, while Ca(2+) inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity.
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PMID:Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. 1757 13

Alpha- and flaviviruses contain class II fusion proteins, which form ion-permeable pores in the target membrane during virus entry. The pores generated during entry of the alphavirus Semliki Forest virus have been shown previously to be blocked by lanthanide ions. Here, analyses of the influence of rare earth ions on the entry of the flaviviruses West Nile virus and Uganda S virus revealed an unexpected effect of lanthanide ions. The results showed that a 30 s treatment of cells with an appropriate lanthanide ion changed the cellular chemistry into a state in which the cells no longer supported the multiplication of flaviviruses. This change occurred in cells treated before, during or after infection, did not inhibit multiplication of Semliki Forest virus and did not interfere with host-cell multiplication. The change was generated in vertebrate and insect cells, and was elicited in the presence of actinomycin D. In vertebrate cells, the change was elicited specifically by La(3+), Ce(3+), Pr(3+) and Nd(3+). In insect cells, additional lanthanide ions had this activity. Further analyses showed that lanthanide ion treatment blocked the ability of the host cell to support the replication of flavivirus RNA. These results open two areas of research: the study of molecular alterations induced by lanthanide ion treatment in uninfected cells and the analysis of the resulting modifications of the flavivirus RNA replicase complex. The findings possibly open the way for the development of a general chemotherapy against flavivirus diseases such as Dengue fever, Japanese encephalitis, West Nile fever and yellow fever.
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PMID:A short treatment of cells with the lanthanide ions La3+, Ce3+, Pr3+ or Nd3+ changes the cellular chemistry into a state in which RNA replication of flaviviruses is specifically blocked without interference with host-cell multiplication. 1794 25

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

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.
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PMID:Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene. 1819 Sep 94

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

Japanese encephalitis (JE) is a serious central nervous system infection and major public health problem in several countries of Southeast Asia including India. This study evaluated the use of IgM ELISA and reverse-transcriptase (RT)-PCR in blood and cerebrospinal fluid (CSF) samples from acute encephalitis patients for the detection of Japanese encephalitis virus (JEV). Forty-four children suffering from acute encephalitis were enrolled, and 36 were selected from whom both CSF and serum samples were available. Twenty-two of the 36 CSF samples were positive for JEV by IgM ELISA and all were negative by RT-PCR. Twenty-three of the 36 serum samples were positive by IgM ELISA while 28 were positive by RT-PCR. Total positivity for JEV infection in CSF and serum samples was 66.7% (24/36) and 83.3% (30/36) respectively by one or both tests. The overall positivity for JEV infection was 86.1% (31/36). We suggest that the use of RT-PCR in serum samples during the early days of JEV infection may be helpful in confirming diagnosis in those cases which are negative for JEV-specific IgM antibodies in both serum and CSF samples.
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PMID:Evaluation of reverse-transcriptase PCR as a diagnostic tool to confirm Japanese encephalitis virus infection. 1924 68

Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.
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PMID:Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein. 1936 2

Japanese encephalitis virus (JEV) is a major cause of acute viral encephalitis in humans. The single-stranded, plus-sense viral genome, which is used for translation and minus-strand RNA synthesis, and its complementary minus-strand viral RNA contain various sequences and RNA secondary structures conserved in flaviviruses, providing potential targets for antisense agents. Here, we investigated the antiviral effects of peptide nucleic acids (PNAs) targeting cis-acting signals at the 5'-untranslated region (UTR), 3'-UTR, and genome cyclization motifs on the plus-strand RNA, as well as the 95-nucleotide 3'-end of the minus-strand RNA, which serves as a template for plus-strand RNA synthesis by the viral RNA-dependent RNA polymerase (RdRp). Among the tested cell-penetrating peptide (CPP)-PNA conjugates, a 17-mer PNA conjugate targeting the top of the 3'-UTR loop structure was most effective in suppressing virus proliferation. In vitro RdRp assays and electrophoretic mobility shift assays using a functional recombinant JEV RdRp showed that the 3'-terminal region-targeting PNAs could inhibit RNA synthesis by competing with viral RdRp for binding to a selected cis-acting element at the 3'-end of plus- and minus-strand viral RNAs. Collectively, our results suggest that CPP-PNA conjugates can suppress JEV proliferation by blocking RNA-protein or RNA-RNA interactions essential for productive viral infection.
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PMID:Inhibition of Japanese encephalitis virus replication by peptide nucleic acids targeting cis-acting elements on the plus- and minus-strands of viral RNA. 1942 3

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive- and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.
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PMID:Heterogeneous nuclear ribonucleoprotein A2 participates in the replication of Japanese encephalitis virus through an interaction with viral proteins and RNA. 2186 91


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