Publications

• mRNA capping by venezuelan equine encephalitis virus nsp1: functional characterization and implications for antiviral research.

  Li C., Guillen J., Rabah N., Blanjoie A., Debart F., Vasseur J.J., Canard B., Decroly E., Coutard B.

  <h4>Unlabelled</h4>Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (Ado ... >> More

  <h4>Unlabelled</h4>Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.<h4>Importance</h4>Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development. << Less

  J. Virol. 89:8292-8303(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase.

  Hooda P., Ishtikhar M., Saraswat S., Bhatia P., Mishra D., Trivedi A., Kulandaisamy R., Aggarwal S., Munde M., Ali N., AlAsmari A.F., Rauf M.A., Inampudi K.K., Sehgal D.

  Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV ... >> More

  Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg²⁺) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg²⁺ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity. << Less

  Molecules 27:0-0(2022) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.

  Ahola T., Kaeaeriaeinen L.

  After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a co ... >> More

  After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule. << Less

  Proc. Natl. Acad. Sci. U.S.A. 92:507-511(1995) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

  Tomar S., Narwal M., Harms E., Smith J.L., Kuhn R.J.

  Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the ... >> More

  Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed α/β structure and is in the folded native conformation. << Less

  Protein Expr Purif 79:277-284(2011) [PubMed] [EuropePMC]