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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel isolate of Seoul (SEO) hantaviruses was detected and identified in Rattus norvegicus in Shandong Province, China and designated as JUN5-14. The partial M segment and the coding region of nucleocapsid protein (NP) in the S segment of JUN5-14 were PCR-amplified and sequenced. Nucleotide sequence analysis of the partial M segment (300 bp) revealed that JUN5-14 isolate was closely related to former SEO isolates in Shandong (97.3-99.0% homology) and non-Shandong SEO viruses (84.1-97.7% homology) but distantly related to other hantaviruses (61.5-75.1% homology). Consistent with the M segment, the coding region of the NP showed 87.5-97.8% and 97.9-99.8% identity with SEO viruses and 55.2-75.8% and 47.2-84.4% homology with other hantaviruses, at nucleotide and amino acid level, respectively. The virus isolate was identified as a member of the subtype 3 (S3) of SEO viruses by phylogenetic trees generated from the nucleotide sequences of the S and M segments. In order to develop a diagnostic assay for hantavirus infection in human, the full-length NP gene of JUN5-14 was expressed in BHK21 cells using the T7 RNA polymerase expression system. The NP expression was confirmed by indirect immunofluorescence assay (IFA) and Western blotting. The expressed NP protein was used as antigen to detect antibody response against NP in patients with hemorrhagic fever with renal syndrome (HFRS) in an IgG-IFA. Sixteen out of seventeen serum samples showed positive for the presence of anti-NP antibodies, indicating that the recombinant NP (rNP) protein of JUN5-14 was a good antigen for detecting hantavirus infection in human.
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PMID:Genetic properties of medium (M) and small (S) genomic RNA segments of Seoul hantavirus isolated from Rattus norvegicus and antigenicity analysis of recombinant nucleocapsid protein. 1692 15

To asses the role of virus load in the pathogenesis of hemorrhagic fever with renal syndrome, the serum Dobrava virus RNA load in 46 patients was measured with a novel quantitative real-time reverse-transcriptase polymerase chain reaction assay and compared to the disease severity. The level of viremia, detected in 26 patients, ranged from 10(2)-10(8) copies/mL of serum. The patients with severe disease had, on average, higher viral RNA loads than patients with a milder course of disease (6.15 vs. 4.67 log(10) copies/mL; P = .053). These results suggest that the Dobrava virus load might be associated with the severity of disease.
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PMID:Dobrava virus RNA load in patients who have hemorrhagic fever with renal syndrome. 1826 19

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.
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PMID:Structure of the Ebola VP35 interferon inhibitory domain. 1912 51

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.
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PMID:Expression of L protein of Hantaan virus 84FLi strain and its application for recovery of minigenomes. 1913 12

Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.
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PMID:The Hantavirus Glycoprotein G1 Tail Contains Dual CCHC-type Classical Zinc Fingers. 1917 34

In this work is presented, for the first time, the expression and purification in a prokaryotic system of the functionally active, recombinant full length VP35 protein of Ebola virus (EBOV). EBOV is an enveloped non-segmented negative-stranded RNA virus belonging to the filovirus family which causes a severe hemorrhagic fever in humans with mortality rates as high as 90%. Several lines of evidence suggest that EBOV interferes with host interferon responses and that the lack of these responses allows its rapidly progressive, overwhelming infection. Recently, the EBOV-encoded VP35 protein, essential cofactor of the viral RNA polymerase complex, has been shown to play an important role as interferon antagonist and the structure of his C-terminal IFN inhibitory domain has been solved. Although it is clearly important to better understand VP35 biochemical functions and its interplay with viral and cellular factors, the attempts to obtain full length E. coli recombinant VP35 (rVP35) have, until now, failed. In this study, we expressed the full length EBOV VP35 in E. coli as a soluble N-terminal His(6)-tag fusion protein and purified it to >95% homogeneity. In order to compare native and rVP35 functions, we characterized the rVP35 for its homo-oligomeric status and its RNA binding capacity showing that bacterially expressed rVP35 has the same properties as VP35 expressed in eukaryotic cells and that, therefore, rVP35 can be used as a valid model for functional studies and the validation of biochemical assays aimed to identify antiviral inhibitors which can interfere with the EBOV replication cycle.
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PMID:Purification and functional characterization of the full length recombinant Ebola virus VP35 protein expressed in E. coli. 1923 84

Several arenaviruses can cause hemorrhagic fever diseases (VHFs) in humans, the pathogenic mechanism of which is poorly understood due to their virulent nature and the lack of molecular clones. A safe, convenient, and economical small animal model of arenavirus hemorrhagic fever is based on guinea pigs infected by the arenavirus Pichinde (PICV). PICV does not cause disease in humans, but an adapted strain of PICV (P18) causes a disease in guinea pigs that mimics arenavirus hemorrhagic fever in humans in many aspects, while a low-passaged strain (P2) remains avirulent in infected animals. In order to identify the virulence determinants within the PICV genome, we developed the molecular clones for both the avirulent P2 and virulent P18 viruses. Recombinant viruses were generated by transfecting plasmids that contain the antigenomic L and S RNA segments of PICV under the control of the T7 promoter into BSRT7-5 cells, which constitutively express T7 RNA polymerase. By analyzing viral growth kinetics in vitro and virulence in vivo, we show that the recombinant viruses accurately recapitulate the replication and virulence natures of their respective parental viruses. Both parental and recombinant virulent viruses led to high levels of viremia and titers in different organs of the infected animals, whereas the avirulent viruses were effectively controlled and cleared by the hosts. These novel infectious clones for the PICV provide essential tools to identify the virulence factors that are responsible for the severe VHF-like disease in infected animals.
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PMID:Development of infectious clones for virulent and avirulent pichinde viruses: a model virus to study arenavirus-induced hemorrhagic fevers. 1938 14

Tensaw virus (TSV) belongs to the genus Orthobunyavirus within the Bunyaviridae family. Although TSV does not cause hemorrhagic fever as some other members of its family, serological studies have shown that serum from Florida residents react against TSV indicating viral infection in humans. In this study, the three RNA genome segments of a TSV isolated from Anopheles crucians mosquitoes collected in North Central Florida in 2006 and a TSV isolate obtained from the CDC, Fort Collins, were sequenced and compared to other Bunyaviridae. The placement of the TSVs within the Bunyamwera serogroup was confirmed by phylogenetic analysis of the inferred amino acid (aa) sequence of proteins coded by each of the RNA segments separately as well as by the combined tree of the same three inferred proteins. The N terminal glycoprotein (Gn) encoded by the M segment contained the 18 conserved Cysteines present in Bunyamwera and California serogroups, the two glycosylation sites, and residues considered potential proteolytic cleavage sites conserved in other Bunyaviridae. The TSV L protein displayed all the strictly conserved amino acids in the four conserved regions known to be catalytically active for the RNA dependent RNA polymerase transcriptase and replicase activities. The amino acid conservation between the two TSV viral isolates was 100, 99.4, and 99.6% for the S, M, and L segments, respectively.
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PMID:Tensaw virus genome sequence and its relation to other Bunyaviridae. 1976 Jan 76

Dengue viruses (DENV) cause the most common arboviral disease afflicting men. Clinical manifestations range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the disease pathogenesis are not fully understood. The severity of the disease seems to be influenced by both viral and host factors. Subgenomic replicons of DENV can be used to study viral replication mechanisms and evaluate the effects of antiviral drugs on viral replication. The objective was to generate and characterize biologically a replicon from a clinical isolate of DENV-3, as part of our studies to understand how this new isolate interacts with cells. To obtain this replicon several RT-PCR fragments encoding the non-structural proteins genes were cloned in high-copy vectors, and used to assemble the replicon in a BAC plasmid vector containing a synthetic DNA molecule encoding the 5' and 3' ends of a viral cDNA with a T7 DNA-dependent RNA polymerase promoter and a ribozyme. In vitro transcribed RNA recovered from this BAC plasmid was transfected into C6/36 mosquito cells, and dengue virus protein expression was assessed by indirect immunofluorescence using polyclonal antibodies. The results showed that the replicon was replicated efficiently in cells, demonstrating successful assembly of a DENV-3 replicon.
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PMID:Construction and characterization of a stable subgenomic replicon system of a Brazilian dengue virus type 3 strain (BR DEN3 290-02). 1976 96

Hantaviruses are rodent-borne viruses capable of causing human disease. The Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome in East Asia. To our knowledge, we report the first domestically acquired case of hemorrhagic fever with renal syndrome caused by the Seoul virus, confirmed by serology testing, reverse-transcriptase polymerase chain reaction, and nucleotide sequence analysis. The patient presented with myalgias and fever, and developed acute renal failure.
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PMID:Domestically acquired seoul virus causing hemorrhagic fever with renal syndrome-Maryland, 2008. 1984


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