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.

• 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.

• Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities.

  Ahola T., Laakkonen P., Vihinen H., Kaeaeriaeinen L.

  The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction ... >> More

  The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covalent complex m7GMP-nsP1. These reactions are virus specific and thus potential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the large alphavirus-like superfamily. Mutations of D64, D90, R93, C135, C142, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1. All MT-negative mutants lost also the GT activity, confirming that methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT reaction without destroying MT activity. Conservation of residues essential for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Residues D64 and D90 were necessary for AdoMet binding, as measured by UV cross-linking. Secondary structure predictions of nsP1 and other proteins of the superfamily place these residues in positions corresponding to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related. << Less

  J. Virol. 71:392-397(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Conventional and unconventional mechanisms for capping viral mRNA.

  Decroly E., Ferron F., Lescar J., Canard B.

  In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral ... >> More

  In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5' ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5'-caps and escape innate immunity. << Less

  Nat. Rev. Microbiol. 10:51-65(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.