50 research outputs found
Flaviviral methyltransferase/RNA interaction: structural basis for enzyme inhibition
No full textFlaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA capping process, transferring a methyl group from S-adenosyl-L-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2'O position of the first nucleotide following the cap guanine (nucleoside-2'O methyltransferase). The RNA capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2'O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor
De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase
No full textAll plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3′ end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3′-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis
Structural bases for substrate recognition and activity in Meaban virus nucleoside-2'-O-methyltransferase
No full textViral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S-adenosyl-L-methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine-N7)-methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside-2'-O-)-methyltransferases (2'OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and purified two constructs of Meaban virus (MV; genus Flavivirus) NS5 protein MTase domain (residues 1-265 and 1-293, respectively). We report here the three-dimensional structure of the shorter MTase construct in complex with the cofactor S-adenosyl-L-methionine, at 2.9 angstrom resolution. Inspection of the refined crystal structure, which highlights structural conservation of specific active site residues, together with sequence analysis and structural comparison with Dengue virus 2'OMTase, suggests that the crystallized enzyme belongs to the 2'OMTase subgroup. Enzymatic assays show that the short MV MTase construct is inactive, but the longer construct expressed can transfer a methyl group to the ribose 2'O atom of a short GpppAC(5) substrate. West Nile virus MTase domain has been recently shown to display both N7 and 2'O MTase activity on a capped RNA substrate comprising the 5'-terminal 190 nt of the West Nile virus genome. The lack of N7 MTase activity here reported for MV MTase may be related either to the small size of the capped RNA substrate, to its sequence, or to different structural properties of the C-terminal regions of West Nile virus and MV MTase-domains
Recognition of the RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus
No full textThe 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
Crystal structure and activity of Kunjin virus NS3 helicase; Protease and helicase domain assembly in the full length NS3 protein
No full textFlaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 angstrom 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. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus. (C) 2007 Elsevier Ltd. All rights reserved
Antiviral activity of the natural alkaloid anisomycin against dengue and Zika viruses
No full textFlaviviruses constitute a public health concern because of their global burden and the lack of specific antiviral treatment. Here we investigated the antiviral activity of the alkaloid anisomycin against dengue (DENV) and Zika (ZIKV) viruses. At non-cytotoxic concentrations, anisomycin strongly inhibited the replication of reference strains and clinical isolates of all DENV serotypes and Asian and African strains of ZIKV in Vero cells. Anisomycin also prevented DENV and ZIKV multiplication in human cell lines. While initial steps of DENV and ZIKV replicative cycle were unaffected, a high inhibition of viral protein expression was demonstrated after treatment with anisomycin. DENV RNA synthesis was strongly reduced in anisomycin treated cultures, but the compounddid not exert a direct inhibitory effect on 2′ O-methyltransferase or RNA polymerase activities of DENV NS5 protein. Furthermore, anisomycin-mediated activation of p38 signaling was not related to the antiviral action ofthe compound. The evaluation of anisomycin efficacy in a mouse model of ZIKV morbidity and mortality revealed that animals treated with a low dose of anisomycin exhibited a significant reduction in viremia levels anddied significantly later than the control group. This protective effect was lost at higher doses, though. In conclusion, anisomycin is a potent and selective in vitro inhibitor of DENV and ZIKV that impairs a post-entry step of viral replication; and a low-dose anisomycin treatment may provide some minimal benefit in a mouse model.Fil: Quintana, Verónica Mara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Selisko, B.. Centre National de la Recherche Scientifique; FranciaFil: Brunetti, Jesús Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Eydoux, C.. Centre National de la Recherche Scientifique; FranciaFil: Guillemot, J.C.. Centre National de la Recherche Scientifique; FranciaFil: Canard, B.. Centre National de la Recherche Scientifique; FranciaFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Julander, J.. State University of Utah; Estados UnidosFil: Castilla, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin
The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core
No full textAbstractThe nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5′-triphosphatase (NTPase), and RNA 5′-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (ΔNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of ΔNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of ΔNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of ΔNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain
Structure and functionality in flavivirus NS-proteins: Perspectives for drug design
No full textFlaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads
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.
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