The Experts below are selected from a list of 114 Experts worldwide ranked by ideXlab platform
Bruno Coutard - One of the best experts on this subject based on the ideXlab platform.
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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
S. S. Baba - One of the best experts on this subject based on the ideXlab platform.
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Preliminary studies on the use of solid-phase immunosorbent techniques for the rapid detection of Wesselsbron Virus (WSLV) IgM by haemagglutination-inhibition
Comparative Immunology Microbiology and Infectious Diseases, 1999Co-Authors: S. S. Baba, A H Fagbami, C.k. OjehAbstract:Abstract Serum samples from 446 randomly selected persons belonging to different age groups and locations in Nigeria were tested for the presence of WSLV IgM using the flaviVirus haemagglutination-inhibition (HI) test adopted to the solid-phase immunosorbent technique (SPIT). 61 (14%) persons had IgM to WSLV only, while 9 (2%) persons had heterologous IgM to WSLV and two other flaviViruses, namely yellow fever and Uganda S Viruses. There was a high prevalence of IgM in people of younger age groups than those in older groups. The majority of the IgM positive sera (67 (96%) of the 70 positive sera reacted to high titres (≥1:80). With the conventional HI tests, 314 (70%) of the total sera tested had HI antibodies to one or more flaviViruses (yellow fever, West Nile, Potiskum, Zika and Uganda S) out of which 305/314 (97%) had antibodies to 3 or more flaviViruses used in the tests. Although SPIT may not be as sensitive as the conventional HI test, it was found to be more specific and could be adopted for the detection of early WSLV infections in flaviVirus hyperendemic environments.
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Wesselsbron Virus antibody in domestic animals in Nigeria: retrospective and prospective studies.
The new microbiologica, 1995Co-Authors: S. S. Baba, A H Fagbami, C.k. OjehAbstract:Retrospective and prospective serological surveys to determine the prevalence of Wesslsbron (WSL) Virus infections in animal populations were carried out in different vegetational zones in Nigeria. Sera from 1,492 animals comprising 292 camels, 81 horses, 4 donkeys, 320 cattle, 235 sheep, 260 goats, 114 pigs, 101 dogs and 85 domestic fowls were assayed by haemagglutination-inhibition (HI) test for presence of antibodies to WSL Virus and other flaviVirus antigens: Yellow Fever (YF), Potiskum (POT), Banzi (BAN), Uganda S (UGS) and West Nile (WN) Viruses. Four hundred and eighty one (32%) of the total sera tested were positive for the presence of flaviVirus antibodies. The prevalence rates among animals varied with species and vegetational zones of the country. The highest prevalence was noted in animals from a swamp forest zone and was higher among camels, horses, donkeys and sheep when compared with goats, pigs and fowls in different zones. Although monotypic reactions with WSL Virus antigen were observed in positive sera, the majority of the WSL Virus positive sera cross-reacted with more than two other flaviVirus antigens. Serological cross-reactions were most extensive in WSL Virus positive horse sera. A ten month sentinel survey among 28 cattle, 68 sheep and 30 goats revealed considerable activity of WSL Virus in Nigeria. Of these, 11 cattle and 12 sheep showed antibody conversion to WSL Virus antigen. None of the goats seroconverted. Although, there are no records of outbreak of WSL disease in Nigeria, this study revealed that WSL Virus is actively circulating among livestock populations in this environment. FlaviVirus nucleotide data are needed for final determination of genetic relatedness in this group of Viruses.
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Wesselsbron Virus haemagglutination: studies with erythrocytes of various animal species at different pH and temperatures
Archives of Virology, 1993Co-Authors: S. S. BabaAbstract:The haemagglutinating (HA) properties of the Nigerian strain of Wesselsbron Virus have been investigated using erythrocytes from a wide range of animals. The results showed that Wesselsbron Virus possesses HA activity when extracted using the sucrose and acetone method. The erythrocytes of goose, horse, donkey, pig, cattle, sheep, goat, monkey, man, rabbit, rat, guinea pig and chicken were agglutinated by Wesselsbron Virus at different pH values (5.75–7.0) and temperatures of 4°C, room (25±2°C) and 37°C. The ability to haemagglutinate fell as pH increased, but the effect of incubation at different temperature was not marked. However, under the conditions of the experiment HA pattern was clearest at 37°C. High HA titres (⩾1:16) were consistently obtained using goose, horse, donkey and human erythrocytes at different temperatures.
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Wesselsbron Virus haemagglutination: studies with erythrocytes of various animal species at different pH and temperatures.
Archives of Virology, 1993Co-Authors: S. S. BabaAbstract:The haemagglutinating (HA) properties of the Nigerian strain of Wesselsbron Virus have been investigated using erythrocytes from a wide range of animals. The results showed that Wesselsbron Virus possesses HA activity when extracted using the sucrose and acetone method. The erythrocytes of goose, horse, donkey, pig, cattle, sheep, goat, monkey, man, rabbit, rat, guinea pig and chicken were agglutinated by Wesselsbron Virus at different pH values (5.75–7.0) and temperatures of 4°C, room (25±2°C) and 37°C.
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Wesselsbron Virus haemagglutination: studies with erythrocytes of various animal species at different pH and temperatures.
Archives of Virology, 1993Co-Authors: S. S. BabaAbstract:The haemagglutinating (HA) properties of the Nigerian strain of Wesselsbron Virus have been investigated using erythrocytes from a wide range of animals. The results showed that Wesselsbron Virus possesses HA activity when extracted using the sucrose and acetone method. The erythrocytes of goose, horse, donkey, pig, cattle, sheep, goat, monkey, man, rabbit, rat, guinea pig and chicken were agglutinated by Wesselsbron Virus at different pH values (5.75-7.0) and temperatures of 4 degrees C, room (25 +/- 2 degrees C) and 37 degrees C. The ability to haemagglutinate fell as pH increased, but the effect of incubation at different temperature was not marked. However, under the conditions of the experiment HA pattern was clearest at 37 degrees C. High HA titres (> or = 1:16) were consistently obtained using goose, horse, donkey and human erythrocytes at different temperatures.
Michela Bollati - One of the best experts on this subject based on the ideXlab platform.
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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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MTases and helicases: a medium-throughput approach to viral protein structures
Acta Crystallographica Section A, 2007Co-Authors: Mario Milani, Eloise Mastrangelo, Michela Bollati, G. Sorrentino, M. BolognesiAbstract:In the context of the European VIZIER project, our lab is involved in the study of flaviviral methyltransferases (MTase) and helicases (Hel). FlaviViruses are enveloped positive-strand RNA Viruses, which code for 3 structural and 7 non-structural (NS) proteins. Among the NS, particularly important are the multifunctional proteins NS3 (protease/helicase) and NS5 (Mtase/RNA-polymerase). Flaviviral Hel participate in RNA replication separating the RNA template and daughter strands. We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin Virus, an Australian variant of the West Nile Virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. In addition, we analyzed the activity of the full-length protein and its structure in solution using small angle X-ray scattering (SAXS). Our results show a strong influence of the NS3 protease domain on the helicase activity that can scarcely be explained in term of domains organization and requires further investigations. MTases are involved in the mRNA capping process, resulting in the transfer of methyl groups from the cofactor S-adenosyl-L-methionine (AdoMet) to a capped RNA substrate. We solved the crystal structures of the Wesselsbron Virus methyltransferase (MTase) in complex with AdoMet and with both the cofactor and the capped substrate GpppG, at 2.0 A and 1.9 A resolution, respectively. Wesselsbron is an African mosquito-borne FlaviVirus belonging to the Yellow Fever Virus group that affects animals and human beings. Comparison of the two structures shows that the presence of GpppG stabilizes the N-terminal subdomain, as indicated by the higher B-factor values relative to the other MTases. In order to further characterize the function and catalytic activity of MTase, assays with different substrates are in progress.
Eloise Mastrangelo - One of the best experts on this subject based on the ideXlab platform.
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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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MTases and helicases: a medium-throughput approach to viral protein structures
Acta Crystallographica Section A, 2007Co-Authors: Mario Milani, Eloise Mastrangelo, Michela Bollati, G. Sorrentino, M. BolognesiAbstract:In the context of the European VIZIER project, our lab is involved in the study of flaviviral methyltransferases (MTase) and helicases (Hel). FlaviViruses are enveloped positive-strand RNA Viruses, which code for 3 structural and 7 non-structural (NS) proteins. Among the NS, particularly important are the multifunctional proteins NS3 (protease/helicase) and NS5 (Mtase/RNA-polymerase). Flaviviral Hel participate in RNA replication separating the RNA template and daughter strands. We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin Virus, an Australian variant of the West Nile Virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. In addition, we analyzed the activity of the full-length protein and its structure in solution using small angle X-ray scattering (SAXS). Our results show a strong influence of the NS3 protease domain on the helicase activity that can scarcely be explained in term of domains organization and requires further investigations. MTases are involved in the mRNA capping process, resulting in the transfer of methyl groups from the cofactor S-adenosyl-L-methionine (AdoMet) to a capped RNA substrate. We solved the crystal structures of the Wesselsbron Virus methyltransferase (MTase) in complex with AdoMet and with both the cofactor and the capped substrate GpppG, at 2.0 A and 1.9 A resolution, respectively. Wesselsbron is an African mosquito-borne FlaviVirus belonging to the Yellow Fever Virus group that affects animals and human beings. Comparison of the two structures shows that the presence of GpppG stabilizes the N-terminal subdomain, as indicated by the higher B-factor values relative to the other MTases. In order to further characterize the function and catalytic activity of MTase, assays with different substrates are in progress.
Mario Milani - One of the best experts on this subject based on the ideXlab platform.
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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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MTases and helicases: a medium-throughput approach to viral protein structures
Acta Crystallographica Section A, 2007Co-Authors: Mario Milani, Eloise Mastrangelo, Michela Bollati, G. Sorrentino, M. BolognesiAbstract:In the context of the European VIZIER project, our lab is involved in the study of flaviviral methyltransferases (MTase) and helicases (Hel). FlaviViruses are enveloped positive-strand RNA Viruses, which code for 3 structural and 7 non-structural (NS) proteins. Among the NS, particularly important are the multifunctional proteins NS3 (protease/helicase) and NS5 (Mtase/RNA-polymerase). Flaviviral Hel participate in RNA replication separating the RNA template and daughter strands. We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin Virus, an Australian variant of the West Nile Virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. In addition, we analyzed the activity of the full-length protein and its structure in solution using small angle X-ray scattering (SAXS). Our results show a strong influence of the NS3 protease domain on the helicase activity that can scarcely be explained in term of domains organization and requires further investigations. MTases are involved in the mRNA capping process, resulting in the transfer of methyl groups from the cofactor S-adenosyl-L-methionine (AdoMet) to a capped RNA substrate. We solved the crystal structures of the Wesselsbron Virus methyltransferase (MTase) in complex with AdoMet and with both the cofactor and the capped substrate GpppG, at 2.0 A and 1.9 A resolution, respectively. Wesselsbron is an African mosquito-borne FlaviVirus belonging to the Yellow Fever Virus group that affects animals and human beings. Comparison of the two structures shows that the presence of GpppG stabilizes the N-terminal subdomain, as indicated by the higher B-factor values relative to the other MTases. In order to further characterize the function and catalytic activity of MTase, assays with different substrates are in progress.
