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      Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities


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          Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg 2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (−) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

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          Global Illness and Deaths Caused by Rotavirus Disease in Children

          To estimate the global illness and deaths caused by rotavirus disease, we reviewed studies published from 1986 to 2000 on deaths caused by diarrhea and on rotavirus infections in children. We assessed rotavirus-associated illness in three clinical settings (mild cases requiring home care alone, moderate cases requiring a clinic visit, and severe cases requiring hospitalization) and death rates in countries in different World Bank income groups. Each year, rotavirus causes approximately 111 million episodes of gastroenteritis requiring only home care, 25 million clinic visits, 2 million hospitalizations, and 352,000–592,000 deaths (median, 440,000 deaths) in children <5 years of age. By age 5, nearly every child will have an episode of rotavirus gastroenteritis, 1 in 5 will visit a clinic, 1 in 60 will be hospitalized, and approximately 1 in 293 will die. Children in the poorest countries account for 82% of rotavirus deaths. The tremendous incidence of rotavirus disease underscores the urgent need for interventions, such as vaccines, to prevent childhood deaths in developing nations.
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            Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

            The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.
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              Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of Nsp2.

              Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

                Author and article information

                J Mol Biol
                J. Mol. Biol
                Journal of Molecular Biology
                29 July 2006
                22 September 2006
                29 July 2006
                : 362
                : 3
                : 539-554
                [1 ]Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, MD 20892, USA
                [2 ]Centro de Bioinformatica y Simulacion Molecular (CBSM), Universidad de Talca, 2 Norte 685, Casilla 721, Talca - Chile
                Author notes
                [* ]Corresponding author. jpatton@ 123456niaid.nih.gov

                Present address: R. Vasquez-Del Carpio, Structural Biology Program, Department of Molecular Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA.


                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 2 June 2006
                : 13 July 2006
                : 20 July 2006

                Molecular biology
                ds, double-stranded,ss, single-stranded,dlp, double-layered particle,rdrp, rna-dependent rna polymerase,ntpase, nucleoside-triphosphate phosphohydrolase,pkci, protein kinase c-interacting protein,hit, histidine triad,rtpase, rna triphosphatase,vls, viroplasm-like structure,tap, tobacco acid pyrophosphatase,rotavirus replication,viroplasm formation,nsp2,rtpase activity,ntpase activity


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