The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Barbara Selisko - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of the nucleoside 2 o methyltransferase activity of dengue virus protein ns5 using purified Capped RNA oligonucleotides 7megpppacn and gpppacn
Journal of General Virology, 2010Co-Authors: Barbara Selisko, Bruno Canard, Frederic Peyrane, Karine Alvarez, Etienne DecrolyAbstract:The flavivirus RNA genome contains a conserved cap-1 structure, 7MeGpppA2′OMeG, at the 5′ end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2′O)-MTases (2′O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2′O-MTase activity of NS5MTase of dengue virus (NS5MTaseDV) using purified, short, Capped RNA substrates (7MeGpppAC n or GpppAC n ). NS5MTaseDV methylated both types of substrate exclusively at the 2′O position. The efficiency of 2′O-methylation did not depend on the methylation of the N7 position. Using 7MeGpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTaseDV 2′O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7MeGpppAC5 and the co-substrate S-adenosyl-l-methionine (AdoMet) were determined (0.39 and 3.26 μM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2′O-MTase activity of NS5MTaseDV showed a low turnover of 3.25×10−4 s−1. Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTaseDV, which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC50 0.34 μM) and sinefungin (IC50 0.63 μM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.
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recognition of RNA cap in the wesselsbron virus ns5 methyltransferase domain implications for RNA capping mechanisms in flavivirus
Journal of Molecular Biology, 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Xavier De Lamballerie, Etienne Decroly, Bruno CoutardAbstract:The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′–5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the Capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.
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Recognition of the RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus
'Elsevier BV', 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Xavier De Lamballerie, Etienne Decroly, Bruno CoutardAbstract:The mRNA-capping process starts with the conversion of a 5V-triphosphate end into a 5V-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5V–5Vphosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-L-methionine to N7-guanine (cap 0) and to the ribose 2VOH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2VO-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-Lmethionine cofactor, S-adenosyl-L-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the Capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate,Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of theWesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7- methyltransferase and 2VO-methyltransferase activities
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high yield production of short gpppa and 7megpppa Capped RNAs and hplc monitoring of methyltransfer reactions at the guanine n7 and adenosine 2 o positions
Nucleic Acids Research, 2007Co-Authors: Frederic Peyrane, Delphine Benarroch, Barbara Selisko, Bruno Canard, Etienne Decroly, Jean-jacques Vasseur, Karine AlvarezAbstract:Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-Capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The Capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced Capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
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an RNA cap nucleoside 2 o methyltransferase in the flavivirus RNA polymerase ns5 crystal structure and functional characterization
The EMBO Journal, 2002Co-Authors: Marie-pierre Egloff, Jean-louis Romette, Delphine Benarroch, Barbara Selisko, Bruno CanardAbstract:Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-l-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 Å resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2′-O-)-methyltransferase activity on Capped RNA. The structure of a teRNAry complex comprising S-adenosyl-l-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.
Etienne Decroly - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of the nucleoside 2 o methyltransferase activity of dengue virus protein ns5 using purified Capped RNA oligonucleotides 7megpppacn and gpppacn
Journal of General Virology, 2010Co-Authors: Barbara Selisko, Bruno Canard, Frederic Peyrane, Karine Alvarez, Etienne DecrolyAbstract:The flavivirus RNA genome contains a conserved cap-1 structure, 7MeGpppA2′OMeG, at the 5′ end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2′O)-MTases (2′O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2′O-MTase activity of NS5MTase of dengue virus (NS5MTaseDV) using purified, short, Capped RNA substrates (7MeGpppAC n or GpppAC n ). NS5MTaseDV methylated both types of substrate exclusively at the 2′O position. The efficiency of 2′O-methylation did not depend on the methylation of the N7 position. Using 7MeGpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTaseDV 2′O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7MeGpppAC5 and the co-substrate S-adenosyl-l-methionine (AdoMet) were determined (0.39 and 3.26 μM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2′O-MTase activity of NS5MTaseDV showed a low turnover of 3.25×10−4 s−1. Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTaseDV, which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC50 0.34 μM) and sinefungin (IC50 0.63 μM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.
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recognition of RNA cap in the wesselsbron virus ns5 methyltransferase domain implications for RNA capping mechanisms in flavivirus
Journal of Molecular Biology, 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Xavier De Lamballerie, Etienne Decroly, Bruno CoutardAbstract:The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′–5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the Capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.
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Recognition of the RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus
'Elsevier BV', 2009Co-Authors: Michela Bollati, Mario Milani, Barbara Selisko, Simona Nonnis, Gabriella Tedeschi, Stefano Ricagno, Eloise Mastrangelo, Xavier De Lamballerie, Etienne Decroly, Bruno CoutardAbstract:The mRNA-capping process starts with the conversion of a 5V-triphosphate end into a 5V-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5V–5Vphosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-L-methionine to N7-guanine (cap 0) and to the ribose 2VOH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2VO-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-Lmethionine cofactor, S-adenosyl-L-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, N7MeGpppG, and N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the Capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate,Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of theWesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7- methyltransferase and 2VO-methyltransferase activities
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high yield production of short gpppa and 7megpppa Capped RNAs and hplc monitoring of methyltransfer reactions at the guanine n7 and adenosine 2 o positions
Nucleic Acids Research, 2007Co-Authors: Frederic Peyrane, Delphine Benarroch, Barbara Selisko, Bruno Canard, Etienne Decroly, Jean-jacques Vasseur, Karine AlvarezAbstract:Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-Capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The Capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced Capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
Amiya K Banerjee - One of the best experts on this subject based on the ideXlab platform.
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histidine mediated RNA transfer to gdp for unique mRNA capping by vesicular stomatitis virus RNA polymerase
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Tomoaki Ogino, Satya P Yadav, Amiya K BanerjeeAbstract:The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the Capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and BoRNA disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.
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histidine mediated RNA transfer to gdp for unique mRNA capping by vesicular stomatitis virus RNA polymerase
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Tomoaki Ogino, Satya P Yadav, Amiya K BanerjeeAbstract:The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the Capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and BoRNA disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.
Ervin Fodor - One of the best experts on this subject based on the ideXlab platform.
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the RNA polymerase of influenza a virus mechanisms of viral transcription and replication
Acta Virologica, 2013Co-Authors: Ervin FodorAbstract:: The influenza A virus RNA genome segments are packaged in ribonucleoprotein complexes containing RNA polymerase and nucleoprotein. The ribonucleoprotein is involved in the transcription of viral genes and replication of the viral RNA genome in the nucleus of the infected cells, and represents the minimal transcriptional and replicative machinery of an influenza virus. During transcription, the viral RNA polymerase synthesizes Capped and polyadenylated mRNA using 5΄ Capped RNA primers. During replication, the viral RNA polymerase generates a complementary RNA (cRNA) replication intermediate, a full-length complement of the vRNA that serves as a template for the synthesis of new copies of vRNA. The nucleoprotein is also an essential component of the viral transcriptional machinery. The molecular determinants of the transcriptional and replicative activities of the viral RNA polymerase are not fully understood, but recent data suggest that transcription is performed by a cis-acting RNA polymerase, forming part of the ribonucleoprotein complex, while replication might be carried out by a trans-acting RNA polymerase. Viral as well as cellular factors are known to be involved in the regulation of the activities of the RNA polymerase, e.g. the viral nuclear export protein has been shown to regulate the accumulation of viral transcription and replication products. The viral transcriptional machinery represents an attractive target for the development of antiviral drugs and lead compounds targeting nucleoprotein and the PA endonuclease domain of the RNA polymerase have already been identified.
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A cluster of conserved basic amino acids near the C-terminus of the PB1 subunit of the influenza virus RNA polymerase is involved in the regulation of viral transcription.
Virology, 2008Co-Authors: Philip S. Kerry, Nicholas Willsher, Ervin FodorAbstract:Synthesis of influenza virus mRNA by the viral RNA polymerase complex is primed by Capped RNA fragments generated by endonuclease cleavage of host pre-mRNA by the polymerase subunit PB1. In previous studies, endonuclease and promoter-binding sites have been described in the C-terminal region of PB1. Here, we have identified an additional region near the C-terminus of PB1 involved in producing Capped RNA primers for viral transcription. In particular, mutations of basic amino acids K669, R670, and R672 inhibited primer-dependent viral mRNA synthesis. In contrast, primer-independent cRNA and vRNA syntheses were only marginally affected. Additionally, recombinant viruses containing the K669A or R672A mutations expressed reduced amounts of mRNA compared to cRNA during infection and were attenuated in cell culture. Further in vitro analysis showed that these mutations inhibited the ability of the polymerase to initiate mRNA synthesis by causing a reduction in binding to the vRNA promoter and Capped RNA. These results suggest that this region plays a critical role in the regulation of viral mRNA transcription.
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functional association between viral and cellular transcription during influenza virus infection
Reviews in Medical Virology, 2006Co-Authors: Othmar G Engelhardt, Ervin FodorAbstract:Influenza viruses replicate and transcribe their segmented negative-sense single-stranded RNA genome in the nucleus of the infected host cell. All RNA synthesising activities associated with influenza virus are performed by the virally encoded RNA-dependent RNA polymerase (RdRp) that consists of three subunits, PA, PB1 and PB2. However, viral transcription is critically dependent on on-going cellular transcription, in particular, on activities associated with the cellular DNA-dependent RNA polymerase II (Pol II). Thus, the viral RdRp uses short 5′ Capped RNA fragments, derived from cellular Pol II transcripts, as primers for viral mRNA synthesis. These Capped RNA primers are generated by cleavage of host Pol II transcripts by an endonuclease activity associated with the viral RdRp. Moreover, some viral transcripts require splicing and since influenza virus does not encode splicing machinery, it is dependent on host splicing, an activity also related to Pol II transcription. Despite these functional links between viral and host Pol II transcription, there has been no evidence that a physical association existed between the two transcriptional machineries. However, recently it was reported that there is a physical interaction between the trimeric viral RdRp and cellular Pol II. The viral RdRp was found to interact with the C-terminal domain (CTD) of initiating Pol II, at a stage in the transcription cycle when capping takes place. It was therefore proposed that this interaction may be required for the viral RNA (vRNA) polymerase to gain access to Capped RNA substrates for endonucleolytic cleavage. The virus not only relies on cellular factors to support its own RNA synthesis, but also subverts cellular pathways in order to generate an environment optimised for viral multiplication. In this respect, the interaction of the viral NS1 protein with factors involved in cellular pre-mRNA processing is of particular relevance. The virus also alters the distribution of Pol II on cellular genes, leading to a reduction in elongating Pol II thereby contributing to the phenomenon known as host shut-off. Copyright © 2006 John Wiley & Sons, Ltd.
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a single amino acid mutation in the pa subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of Capped RNAs
Journal of Virology, 2002Co-Authors: Ervin Fodor, Mandy Crow, Louise J Mingay, Tao Deng, Jane Sharps, Pierre Fechter, George G BrownleeAbstract:The influenza A virus RNA-dependent RNA polymerase consists of three subunits—PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting Capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA→cRNA→vRNA), but its transcriptional activity (vRNA→mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, Capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.
Sean P J Whelan - One of the best experts on this subject based on the ideXlab platform.
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ribose 2 o methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine n 7 methylation by the large polymerase protein
Journal of Virology, 2009Co-Authors: Amal A Rahmeh, Jianrong Li, Philip J Kranzusch, Sean P J WhelanAbstract:Nonsegmented negative-strand (NNS) RNA viruses use a common strategy to express their genomes (for a review, see reference 33). Their genomic RNA is encapsidated by the viral nucleocapsid (N) protein, and it is this N-RNA complex that serves as the template for the viral RNA-dependent RNA polymerase (RdRP). The RdRP comprises a virus-encoded large (L) polymerase protein that possesses all of the enzymatic activities necessary for the synthesis, capping, and polyadenylation of the mRNA and the replication of the genome. The L protein requires an additional cofactor, a phosphoprotein (P), which is required for template recognition. Some NNS RNA viruses require additional viral proteins for authentic RNA synthesis, but those proteins do not appear to contain catalytic activities. Vesicular stomatitis virus (VSV), the prototype of the family Rhabdoviridae, has long served as a model to understand RNA synthesis in the NNS RNA viruses. Purified VSV particles synthesize mRNA in vitro (3), and this can also be accomplished by using a recombinant N-RNA template purified from virus supplemented with recombinant L (rL) and rP (19, 22). During mRNA synthesis, the RdRP initiates synthesis at a 3′-proximal site to copy the viral genes in a polar and sequential manner (1, 2). In response to a specific promoter element, the RdRP initiates mRNA synthesis at a highly conserved gene start sequence, which for VSV is 3′-UUGUCNNUAG-5′ (21, 34, 35), and recognizes the cognate element in the nascent transcript to add an mRNA cap structure (23, 30, 31). Termination of mRNA synthesis is also controlled such that the VSV RdRP recognizes the sequence 3′-AUACUUUUUUU-5′ to polyadenylate the mRNA through reiterative transcription of the U tract and to terminate synthesis of the mRNA (4, 5, 14). The mechanism by which the VSV L protein adds the mRNA cap structure is distinct from that of all other known capping reactions. Specifically, a polyribonucelotidyltransferase (PRNTase) transfers a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate (23). This is in contrast to other capping reactions, in which a guanylyltransferase transfers GMP derived from GTP onto a diphosphate acceptor RNA (for a review, see reference 10). It is generally thought that the capping activity of L resides within one of six conserved regions (CR) that were identified by sequence alignments (26). Amino acid substitutions in a conserved GXXT(X68-70)HR motif in CRV prevent mRNA cap addition, implicating CRV as the PRNTase (19). In addition to the altered mechanism of cap addition, methylation of the cap structure is also unusual in that both guanine-N-7 (G-N-7) and ribose 2′-O positions are modified via what appears to be a single methyltransferase (MTase) domain within the L protein (11, 12, 17, 20). Sequence alignments and structural predictions between known 2′-O MTases and CRVI of L identified a putative MTase domain comprising the catalytic tetrad K-D-K-E and a GxGxG binding site for the methyl donor S-adenosyl-l-methionine (SAM) (6, 9). In transcription reactions carried out using rVSV, single amino acid substitutions in this K-D-K-E motif ablate all mRNA cap methylation (11, 12, 17), whereas single amino acid substitutions within the predicted SAM binding motif either prevented all cap methylation or specifically reduced G-N-7 methylation (20). These results suggested that the two methylase activities share a single binding site for SAM and that the two reactions can proceed in an unconventional order in which 2′-O methylation occurs first. However, those experiments could not determine whether the K-D-K-E motif is specifically required for G-N-7 methylation, as the lack of cap methylation may reflect a requirement for 2′-O methylation to occur first. In contrast to that idea, experiments with Sendai virus (SeV) demonstrated that purified rL protein or a C-terminal fragment comprising CRV and CRVI or CRVI alone was capable of G-N-7 methylation of virus-specific mRNA (24). However, those experiments did not detect a ribose 2′-O methylase activity associated with the SeV L protein and therefore could not address whether the two MTase activities reside within the same region of L. In the present study, we evaluated how the two MTase activities of the VSV L protein are regulated. We reconstituted methylation in vitro by using highly purified rL and Capped RNA. This recapitulated both MTase activities independently of ongoing RNA transcription and allowed us to determine whether the methylase activities of L are coordinated. The results of this study demonstrate that the L protein functions as an efficient 2′-O MTase that facilitates a relatively inefficient G-N-7 MTase and that the K-D-K-E catalytic tetrad is essential for 2′-O methylation but also plays a role in G-N-7 methylation. We further demonstrate that the MTase activities are sequence specific and that they require a longer RNA substrate for methylation than does the PRNTase activity of L in vitro.
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ribose 2 o methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine n 7 methylation by the large polymerase protein
Journal of Virology, 2009Co-Authors: Amal A Rahmeh, Philip J Kranzusch, Sean P J WhelanAbstract:Nonsegmented negative-strand (NNS) RNA viruses use a common strategy to express their genomes (for a review, see reference 33). Their genomic RNA is encapsidated by the viral nucleocapsid (N) protein, and it is this N-RNA complex that serves as the template for the viral RNA-dependent RNA polymerase (RdRP). The RdRP comprises a virus-encoded large (L) polymerase protein that possesses all of the enzymatic activities necessary for the synthesis, capping, and polyadenylation of the mRNA and the replication of the genome. The L protein requires an additional cofactor, a phosphoprotein (P), which is required for template recognition. Some NNS RNA viruses require additional viral proteins for authentic RNA synthesis, but those proteins do not appear to contain catalytic activities. Vesicular stomatitis virus (VSV), the prototype of the family Rhabdoviridae, has long served as a model to understand RNA synthesis in the NNS RNA viruses. Purified VSV particles synthesize mRNA in vitro (3), and this can also be accomplished by using a recombinant N-RNA template purified from virus supplemented with recombinant L (rL) and rP (19, 22). During mRNA synthesis, the RdRP initiates synthesis at a 3′-proximal site to copy the viral genes in a polar and sequential manner (1, 2). In response to a specific promoter element, the RdRP initiates mRNA synthesis at a highly conserved gene start sequence, which for VSV is 3′-UUGUCNNUAG-5′ (21, 34, 35), and recognizes the cognate element in the nascent transcript to add an mRNA cap structure (23, 30, 31). Termination of mRNA synthesis is also controlled such that the VSV RdRP recognizes the sequence 3′-AUACUUUUUUU-5′ to polyadenylate the mRNA through reiterative transcription of the U tract and to terminate synthesis of the mRNA (4, 5, 14). The mechanism by which the VSV L protein adds the mRNA cap structure is distinct from that of all other known capping reactions. Specifically, a polyribonucelotidyltransferase (PRNTase) transfers a monophosphate RNA onto a GDP acceptor through a covalent L-RNA intermediate (23). This is in contrast to other capping reactions, in which a guanylyltransferase transfers GMP derived from GTP onto a diphosphate acceptor RNA (for a review, see reference 10). It is generally thought that the capping activity of L resides within one of six conserved regions (CR) that were identified by sequence alignments (26). Amino acid substitutions in a conserved GXXT(X68-70)HR motif in CRV prevent mRNA cap addition, implicating CRV as the PRNTase (19). In addition to the altered mechanism of cap addition, methylation of the cap structure is also unusual in that both guanine-N-7 (G-N-7) and ribose 2′-O positions are modified via what appears to be a single methyltransferase (MTase) domain within the L protein (11, 12, 17, 20). Sequence alignments and structural predictions between known 2′-O MTases and CRVI of L identified a putative MTase domain comprising the catalytic tetrad K-D-K-E and a GxGxG binding site for the methyl donor S-adenosyl-l-methionine (SAM) (6, 9). In transcription reactions carried out using rVSV, single amino acid substitutions in this K-D-K-E motif ablate all mRNA cap methylation (11, 12, 17), whereas single amino acid substitutions within the predicted SAM binding motif either prevented all cap methylation or specifically reduced G-N-7 methylation (20). These results suggested that the two methylase activities share a single binding site for SAM and that the two reactions can proceed in an unconventional order in which 2′-O methylation occurs first. However, those experiments could not determine whether the K-D-K-E motif is specifically required for G-N-7 methylation, as the lack of cap methylation may reflect a requirement for 2′-O methylation to occur first. In contrast to that idea, experiments with Sendai virus (SeV) demonstrated that purified rL protein or a C-terminal fragment comprising CRV and CRVI or CRVI alone was capable of G-N-7 methylation of virus-specific mRNA (24). However, those experiments did not detect a ribose 2′-O methylase activity associated with the SeV L protein and therefore could not address whether the two MTase activities reside within the same region of L. In the present study, we evaluated how the two MTase activities of the VSV L protein are regulated. We reconstituted methylation in vitro by using highly purified rL and Capped RNA. This recapitulated both MTase activities independently of ongoing RNA transcription and allowed us to determine whether the methylase activities of L are coordinated. The results of this study demonstrate that the L protein functions as an efficient 2′-O MTase that facilitates a relatively inefficient G-N-7 MTase and that the K-D-K-E catalytic tetrad is essential for 2′-O methylation but also plays a role in G-N-7 methylation. We further demonstrate that the MTase activities are sequence specific and that they require a longer RNA substrate for methylation than does the PRNTase activity of L in vitro.
