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)

Avian influenza viruses pose a serious pandemic threat to humans. Better knowledge on cross-species adaptation is important. This study examined the replication and transcription efficiency of ribonucleoprotein complexes reconstituted by plasmid co-transfection between H5N1, H1N1pdm09 and H3N2 influenza A viruses, and to identify mutations in the RNA polymerase subunit that affect human adaptation. Viral RNA polymerase subunits PB1, PB2, PA and NP derived from influenza viruses were co-expressed with pPolI-vNP-Luc in human cells, and with its function evaluated by luciferase reporter assay. A quantitative RT-PCR was used to measure vRNA, cRNA, and mRNA levels for assessing the replication and transcription efficiency. Mutations in polymerase subunit were created to identify signature of increased human adaptability. H5N1 ribonucleoprotein complexes incorporated with PB2 derived from H1N1pdm09 and H3N2 viruses increased the polymerase activity in human cells. Furthermore, single amino acid substitutions at PB2 of H5N1 could affect polymerase activity in a temperature-dependent manner. By using a highly sensitive quantitative reverse transcription-polymerase chain reaction, an obvious enhancement in replication and transcription activities of ribonucleoproteins was observed by the introduction of lysine at residue 627 in the H5N1 PB2 subunit. Although less strongly in polymerase activity, E158G mutation appeared to alter the accumulation of H5N1 RNA levels in a temperature-dependent manner, suggesting a temperature-dependent mechanism in regulating transcription and replication exists. H5N1 viruses can adapt to humans either by acquisition of PB2 from circulating human-adapted viruses through reassortment, or by mutations at critical sites in PB2. This information may help to predict the pandemic potential of newly emerged influenza strains, and provide a scientific basis for stepping up surveillance measures and vaccine production.
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PMID:Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses. 2375 Feb 26

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C60) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C60 fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.
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PMID:Anti-influenza activity of c60 fullerene derivatives. 2378 93

Endonucleases catalyze critical steps in the processing, function, and turnover of many cellular RNAs. It is, therefore, not surprising that a number of viruses encode endonucleases that play important roles in viral gene expression. The virion host shutoff (Vhs) endonuclease of herpes simplex virus, the SOX protein of Kaposi Sarcoma Herpesvirus (KSHV), and the influenza virus PB1 endonuclease have well-characterized functions that stem from their abilities to cleave RNA. Vhs accelerates turnover of many cellular and viral mRNAs, redirecting the cell from host to viral gene expression, counteracting key elements of the innate immune response, and facilitating sequential expression of different classes of viral genes. SOX reduces synthesis of many host proteins during the lytic phase of KSHV infections. PB1 is a component of the influenza RNA polymerase that snatches capped oligonucleotides from cellular pre-mRNAs to serve as primers during viral mRNA synthesis. However, all three proteins have important second functions. Vhs stimulates translation of the 3' cistron of bicistronic mRNAs that have selected cellular internal ribosome entry sites, and stimulates polysome loading and translation of selected viral mRNAs at late times during productive infections. SOX has an alkaline exonuclease activity that is important for processing and maturation of newly synthesized copies of the KSHV genome. The influenza RNA polymerase binds the cap and 5' region of viral mRNAs and recruits eIF4G and other factors to viral mRNAs, allowing them to be translated under conditions of reduced eIF4E functionality. This review will discuss the novel and expected functions of these viral endonucleases.
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PMID:Virus-encoded endonucleases: expected and novel functions. 2390 Sep 73

The influenza virus RNA polymerase, composed of the PB1, PB2 and PA subunits, has a potential role in influencing genetic reassortment. Recent studies on the reassortment of human H3N2 strains suggest that the co-incorporation of PB2 and PA from the same H3N2 strain appears to be important for efficient virus replication; however, the underlying mechanism remains unclear. Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes and demonstrated that the RNP activity was severely impaired when the PA subunit of H3N2 strain A/NT/60/1968 (NT PA) was introduced into H1N1 or H5N1 polymerase. The NT PA did not affect the correct assembly of the polymerase trimeric complex, but it significantly reduced replication-initiation activity when provided with a vRNA promoter and severely impaired the accumulation of RNP, which led to the loss of RNP activity. Mutational analysis demonstrated that PA residues 184N and 383N were the major determinants of the inhibitory effect of NT PA and 184N/383N sequences were unique to human H3N2 strains. Significantly, NT PB2 specifically relieved the inhibitory effect of NT PA, and the PB2 residue 627K played a key role. Our results suggest that PB2 from the same H3N2 strain might be required for overcoming the inhibitory effect of H3N2 PA in the genetic reassortment of influenza virus.
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PMID:Co-incorporation of the PB2 and PA polymerase subunits from human H3N2 influenza virus is a critical determinant of the replication of reassortant ribonucleoprotein complexes. 2393 81

The RNA polymerase of Influenza A virus (IAV), which is comprised of three units PA, PB1 and PB2, is involved in transcription and replication of the influenza virus. In order to develop effective treatment for IAV, researchers have focused on designing drugs targeting IAV polymerase. Currently, crystal structures of the IAV polymerase PA-PB1, PB1-PB2 complexes and the PA subunit have been obtained by several groups, providing useful information regarding potential binding sites in drug design. However, to gain full understanding of the molecular mechanism of IAV polymerase in viral transcription and replication, thereby aiding drug development, a complete atomistic structure of the RNA polymerase is required. In this paper, we employed computer-aided drug design tools to describe the complete structure of the RNA polymerase and proposed a putative mechanism. We predict that the combination of Vancomycin and Oseltamivir will be an effective drug to universally treat IAVs with no resultant drug resistance if this putative mechanism is true.
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PMID:Exploring the molecular mechanism of action between drug and RNA polymerase based on partially-resolved spatial structures. 2413 96

The influenza RNA polymerase complex, which consists of the three subunits PA, PB1, and PB2, is a promising target for the development of new antiviral drugs. A large library of benzofurazan compounds was synthesized and assayed against influenza virus A/WSN/33 (H1N1). Most of the new derivatives were found to act by inhibiting the viral RNA polymerase complex through disruption of the complex formed between subunits PA and PB1. Docking studies were also performed to elucidate the binding mode of benzofurazans within the PB1 binding site in PA and to identify amino acids involved in their mechanism of action. The predicted binding pose is fully consistent with the biological data and lays the foundation for the rational development of more effective PA-PB1 inhibitors.
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PMID:The fight against the influenza A virus H1N1: synthesis, molecular modeling, and biological evaluation of benzofurazan derivatives as viral RNA polymerase inhibitors. 2428 96

The viral RNA-dependent RNA polymerase has been found to contribute to efficient replication in mammalian systems and to the high pathogenicity of H5N1 influenza A virus in humans and other mammals. The terminal untranslated regions of the viral segments perform functions such as polyadenylation and contain signals for genomic packaging and initiation of RNA synthesis. These sequences are highly conserved, apart from a U/C polymorphism at position 4 of the 3' end, most often seen in the polymerase gene segments. However, no study has yet tested whether the untranslated regions of H5N1 make any contribution to its high pathogenicity. Herein, the association of the fourth nucleotide at the 3' end of the untranslated region in segment 2 (PB1), of A/Vietnam/1194/2004 (H5N1), with pathogenicity was examined in mice. To this end, an RNA polymerase reporter system was constructed, and viruses with mutations at this site were rescued. Results showed the U4 in PB1 was found to contribute to greater amounts of RNA-dependent RNA polymerase activity and differentially regulate genomic transcription and replication. Although a recombinant H5N1 virus with the rarer C4 sequence in all eight segments was viable and replicated to high titers in vitro, replacing a single U4 at the 3' termini of the PB1 gene segment enhanced viral reproduction and more pathogenesis. In this way, these data showed the importance of untranslated regions of H5N1 influenza virus to pathogenicity.
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PMID:U4 at the 3' UTR of PB1 segment of H5N1 influenza virus promotes RNA polymerase activity and contributes to viral pathogenicity. 2467 59

Influenza RNA polymerase is composed of three subunits, PA, PB1, and PB2, which interact with each other for transcription and replication of the viral RNA genome in the nucleus of infected cells. PB2 RNA-binding 627-domain (residues 535-693), located in the C-terminus, presents a highly basic surface around residue lysine 627 and has been proposed to interact with viral or cellular factors, resulting in host adaptation. However, the function of this domain is not yet characterized in detail. In this study, we identified RNA-binding activity and RNA-binding surfaces in both the N-terminal and basic C-terminal regions of PB2 627-domain using NMR experiments. Through mutagenesis studies, we confirmed which residues directly interact with RNA and mapped their locations on the RNA-binding surface. In addition, by luciferase activity assays, we showed that influenza virus polymerase activity may correlate with the interaction between PB2 and RNA. Representative host adaptive mutations (residues 591 and 627) were found to be located on the RNA-binding surface and were confirmed to directly interact with RNA and to affect polymerase activity. From these results, we suggest that influenza virus polymerase activity may be regulated through the interaction between PB2 627-domain and RNA and that consequently host adaptation of the virus may be influenced.
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PMID:Biophysical characterization of sites of host adaptive mutation in the influenza A virus RNA polymerase PB2 RNA-binding domain. 2487 50

Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.
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PMID:Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing. 2494 53

The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
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PMID:A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. 2506 5


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