{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T07:13:55Z","timestamp":1791530035257,"version":"4.3.4"},"reference-count":43,"publisher":"Microbiology Society","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2006,1,1]]},"abstract":"<jats:p>A stem\u2013loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a <jats:italic>cis<\/jats:italic>-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication <jats:italic>in vivo<\/jats:italic> and abolished uridylylation of VPg <jats:italic>in vitro<\/jats:italic>. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R<jats:sub>1<\/jats:sub>NNNAAR<jats:sub>2<\/jats:sub>NNNNNNR<jats:sub>3<\/jats:sub>, is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an <jats:italic>in vitro<\/jats:italic> translation\/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (A<jats:sub>5<\/jats:sub>G) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.<\/jats:p>","DOI":"10.1099\/vir.0.81297-0","type":"journal-article","created":{"date-parts":[[2005,12,16]],"date-time":"2005-12-16T18:12:49Z","timestamp":1134756769000},"page":"103-113","source":"Crossref","is-referenced-by-count":74,"title":["Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis"],"prefix":"10.1099","volume":"87","author":[{"given":"Mark J. M.","family":"van Ooij","sequence":"first","affiliation":[{"name":"Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorothee A.","family":"Vogt","sequence":"additional","affiliation":[{"name":"University of California, San Francisco, Mission Bay Genentech Hall, UCSF Department of Microbiology, 600 16th Street, PO Box 2280, San Francisco, CA 94143, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aniko","family":"Paul","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Castro","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Judith","family":"Kuijpers","sequence":"additional","affiliation":[{"name":"Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frank J. M.","family":"van Kuppeveld","sequence":"additional","affiliation":[{"name":"Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig E.","family":"Cameron","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eckard","family":"Wimmer","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raul","family":"Andino","sequence":"additional","affiliation":[{"name":"University of California, San Francisco, Mission Bay Genentech Hall, UCSF Department of Microbiology, 600 16th Street, PO Box 2280, San Francisco, CA 94143, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Willem J. G.","family":"Melchers","sequence":"additional","affiliation":[{"name":"Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"345","reference":[{"key":"R1","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1016\/0092-8674(90)90170-J","article-title":"A functional ribonucleoprotein complex forms around the 5\u2032 end of poliovirus RNA","volume":"63","author":"Andino","year":"1990","journal-title":"Cell"},{"key":"R2","doi-asserted-by":"crossref","first-page":"5516","DOI":"10.1128\/JVI.69.9.5516-5527.1995","article-title":"Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA","volume":"69","author":"Barton","year":"1995","journal-title":"J Virol"},{"key":"R3","doi-asserted-by":"crossref","first-page":"1439","DOI":"10.1093\/emboj\/20.6.1439","article-title":"5\u2032 cloverleaf in poliovirus RNA is a cis -acting replication element required for negative-strand synthesis","volume":"20","author":"Barton","year":"2001","journal-title":"EMBO J"},{"key":"R4","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1038\/ng1583","article-title":"Trans -dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses","volume":"37","author":"Crowder","year":"2005","journal-title":"Nat Genet"},{"key":"R5","doi-asserted-by":"crossref","first-page":"2293","DOI":"10.1101\/gad.12.15.2293","article-title":"Switch from translation to RNA replication in a positive-stranded RNA virus","volume":"12","author":"Gamarnik","year":"1998","journal-title":"Genes Dev"},{"key":"R6","doi-asserted-by":"crossref","first-page":"10979","DOI":"10.1128\/JVI.75.22.10979-10990.2001","article-title":"Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis -replicating element in the coding sequence of 2Apro","volume":"75","author":"Gerber","year":"2001","journal-title":"J Virol"},{"key":"R7","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1006\/prep.1999.1100","article-title":"Production of \u201cauthentic\u201d poliovirus RNA-dependent RNA polymerase (3Dpol) by ubiquitin\u2013protease-mediated cleavage in Escherichia coli","volume":"17","author":"Gohara","year":"1999","journal-title":"Protein Expr Purif"},{"key":"R8","doi-asserted-by":"crossref","first-page":"4590","DOI":"10.1128\/JVI.74.10.4590-4600.2000","article-title":"Identification of a cis -acting replication element within the poliovirus coding region","volume":"74","author":"Goodfellow","year":"2000","journal-title":"J Virol"},{"key":"R9","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1261\/rna.2950603","article-title":"Structure and function analysis of the poliovirus cis -acting replication element (CRE)","volume":"9","author":"Goodfellow","year":"2003a","journal-title":"RNA"},{"key":"R10","doi-asserted-by":"crossref","first-page":"2359","DOI":"10.1099\/vir.0.19132-0","article-title":"The poliovirus 2C cis -acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes","volume":"84","author":"Goodfellow","year":"2003b","journal-title":"J Gen Virol"},{"key":"R11","doi-asserted-by":"crossref","first-page":"6394","DOI":"10.1128\/JVI.74.14.6394-6400.2000","article-title":"Poliovirus requires a precise 5\u2032 end for efficient positive-strand RNA synthesis","volume":"74","author":"Herold","year":"2000","journal-title":"J Virol"},{"key":"R12","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/S1097-2765(01)00205-2","article-title":"Poliovirus RNA replication requires genome circularization through a protein\u2013protein bridge","volume":"7","author":"Herold","year":"2001","journal-title":"Mol Cell"},{"key":"R13","doi-asserted-by":"crossref","first-page":"1573","DOI":"10.1128\/JVI.64.4.1573-1583.1990","article-title":"Complete nucleotide sequence of infectious coxsackievirus B3 cDNA: two initial 5\u2032 uridine residues are regained during plus-strand RNA synthesis","volume":"64","author":"Klump","year":"1990","journal-title":"J Virol"},{"key":"R14","doi-asserted-by":"crossref","first-page":"11560","DOI":"10.1073\/pnas.96.20.11560","article-title":"A coding RNA sequence acts as a replication signal in cardioviruses","volume":"96","author":"Lobert","year":"1999","journal-title":"Proc Natl Acad Sci U S A"},{"key":"R15","doi-asserted-by":"crossref","first-page":"10696","DOI":"10.1128\/JVI.75.22.10696-10708.2001","article-title":"Poliovirus 5\u2032-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis","volume":"75","author":"Lyons","year":"2001","journal-title":"J Virol"},{"key":"R16","doi-asserted-by":"crossref","first-page":"9686","DOI":"10.1128\/JVI.76.19.9686-9694.2002","article-title":"Identification and characterization of a cis -acting replication element ( cre ) adjacent to the internal ribosome entry site of foot-and-mouth disease virus","volume":"76","author":"Mason","year":"2002","journal-title":"J Virol"},{"key":"R17","doi-asserted-by":"crossref","first-page":"1569","DOI":"10.1017\/S1355838298981006","article-title":"The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication","volume":"4","author":"McKnight","year":"1998","journal-title":"RNA"},{"key":"R18","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1128\/JVI.71.1.686-696.1997","article-title":"Kissing of the two predominant hairpin loops in the coxsackie B virus 3\u2032 untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis","volume":"71","author":"Melchers","year":"1997","journal-title":"J Virol"},{"key":"R19","doi-asserted-by":"crossref","first-page":"976","DOI":"10.1017\/S1355838200000480","article-title":"Cross-talk between orientation-dependent recognition determinants of a complex control RNA element, the enterovirus ori R","volume":"6","author":"Melchers","year":"2000","journal-title":"RNA"},{"key":"R20","doi-asserted-by":"crossref","first-page":"9900","DOI":"10.1128\/JVI.76.19.9900-9909.2002","article-title":"Biological significance of a human enterovirus B-specific RNA element in the 3\u2032 nontranslated region","volume":"76","author":"Merkle","year":"2002","journal-title":"J Virol"},{"key":"R21","doi-asserted-by":"crossref","first-page":"2363","DOI":"10.1128\/JVI.71.3.2363-2370.1997","article-title":"An RNA tertiary structure in the 3\u2032 untranslated region of enteroviruses is necessary for efficient replication","volume":"71","author":"Mirmomeni","year":"1997","journal-title":"J Virol"},{"key":"R22","doi-asserted-by":"crossref","first-page":"1647","DOI":"10.1126\/science.1661029","article-title":"Cell-free, de novo synthesis of poliovirus","volume":"254","author":"Molla","year":"1991","journal-title":"Science"},{"key":"R23","doi-asserted-by":"crossref","first-page":"5136","DOI":"10.1128\/JVI.77.9.5136-5144.2003","article-title":"Poliovirus cre (2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis","volume":"77","author":"Morasco","year":"2003","journal-title":"J Virol"},{"key":"R24","doi-asserted-by":"crossref","first-page":"4739","DOI":"10.1128\/JVI.77.8.4739-4750.2003","article-title":"Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis","volume":"77","author":"Murray","year":"2003","journal-title":"J Virol"},{"key":"R25","doi-asserted-by":"crossref","first-page":"7698","DOI":"10.1128\/JVI.79.12.7698-7706.2005","article-title":"Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro","volume":"79","author":"Nayak","year":"2005","journal-title":"J Virol"},{"key":"R26","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1038\/268208a0","article-title":"The location of the polio genome protein in viral RNAs and its implication for RNA synthesis","volume":"268","author":"Nomoto","year":"1977a","journal-title":"Nature"},{"key":"R27","doi-asserted-by":"crossref","first-page":"5345","DOI":"10.1073\/pnas.74.12.5345","article-title":"The 5\u2032-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg","volume":"74","author":"Nomoto","year":"1977b","journal-title":"Proc Natl Acad Sci U S A"},{"key":"R28","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1038\/30529","article-title":"Protein-primed RNA synthesis by purified poliovirus RNA polymerase","volume":"393","author":"Paul","year":"1998","journal-title":"Nature"},{"key":"R29","doi-asserted-by":"crossref","first-page":"10359","DOI":"10.1128\/JVI.74.22.10359-10370.2000","article-title":"Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg","volume":"74","author":"Paul","year":"2000","journal-title":"J Virol"},{"key":"R30","doi-asserted-by":"crossref","first-page":"43951","DOI":"10.1074\/jbc.M307441200","article-title":"A \u201cslide-back\u201d mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus","volume":"278","author":"Paul","year":"2003","journal-title":"J Biol Chem"},{"key":"R31","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1128\/JVI.27.2.357-365.1978","article-title":"Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA","volume":"27","author":"Pettersson","year":"1978","journal-title":"J Virol"},{"key":"R32","doi-asserted-by":"crossref","first-page":"5428","DOI":"10.1002\/j.1460-2075.1996.tb00926.x","article-title":"Cis -element, oriR , involved in the initiation of (\u2212) strand poliovirus RNA: a quasi-globular multi-domain RNA structure maintained by tertiary (\u2018kissing\u2019) interactions","volume":"15","author":"Pilipenko","year":"1996","journal-title":"EMBO J"},{"key":"R33","first-page":"493","article-title":"A simple method of estimating fifty percent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am J Hyg"},{"key":"R34","doi-asserted-by":"crossref","first-page":"10371","DOI":"10.1128\/JVI.74.22.10371-10380.2000","article-title":"Genetic and biochemical studies of poliovirus cis -acting replication element cre in relation to VPg uridylylation","volume":"74","author":"Rieder","year":"2000","journal-title":"J Virol"},{"key":"R35","doi-asserted-by":"crossref","first-page":"12688","DOI":"10.1073\/pnas.0403079101","article-title":"High-resolution structure of a picornaviral internal cis-acting RNA replication element ( cre )","volume":"101","author":"Thiviyanathan","year":"2004","journal-title":"Proc Natl Acad Sci U S A"},{"key":"R36","doi-asserted-by":"crossref","first-page":"7782","DOI":"10.1128\/JVI.69.12.7782-7790.1995","article-title":"Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis -acting function in viral RNA synthesis","volume":"69","author":"van Kuppeveld","year":"1995","journal-title":"J Virol"},{"key":"R37","doi-asserted-by":"crossref","first-page":"3519","DOI":"10.1093\/emboj\/16.12.3519","article-title":"Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release","volume":"16","author":"van Kuppeveld","year":"1997","journal-title":"EMBO J"},{"key":"R38","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1093\/nar\/27.2.485","article-title":"Structural requirements of the higher order RNA kissing element in the enteroviral 3\u2032UTR","volume":"27","author":"Wang","year":"1999","journal-title":"Nucleic Acids Res"},{"key":"R39","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1146\/annurev.ge.27.120193.002033","article-title":"Genetics of poliovirus","volume":"27","author":"Wimmer","year":"1993","journal-title":"Annu Rev Genet"},{"key":"R40","doi-asserted-by":"crossref","first-page":"5079","DOI":"10.1093\/nar\/29.24.5079","article-title":"Conserved RNA secondary structures in Picornaviridae genomes","volume":"29","author":"Witwer","year":"2001","journal-title":"Nucleic Acids Res"},{"key":"R41","doi-asserted-by":"crossref","first-page":"7485","DOI":"10.1128\/JVI.76.15.7485-7494.2002","article-title":"Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis -acting replication element ( cre ) of human rhinovirus type 14","volume":"76","author":"Yang","year":"2002","journal-title":"J Virol"},{"key":"R42","doi-asserted-by":"crossref","first-page":"5152","DOI":"10.1128\/JVI.77.9.5152-5166.2003","article-title":"Functional dissection of a poliovirus cis -acting replication element [PV- cre (2C)]: analysis of single- and dual- cre viral genomes and proteins that bind specifically to PV- cre RNA","volume":"77","author":"Yin","year":"2003","journal-title":"J Virol"},{"key":"R43","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/978-94-011-4485-8_2","article-title":"Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide","volume-title":"In RNA Biochemistry and Biotechnology","author":"Zuker","year":"1999"}],"container-title":["Journal of General Virology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/jgv\/10.1099\/vir.0.81297-0?crawler=true","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,10,6]],"date-time":"2020-10-06T12:32:20Z","timestamp":1601987540000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.microbiologyresearch.org\/content\/journal\/jgv\/10.1099\/vir.0.81297-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2006,1,1]]},"references-count":43,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2006,1,1]]}},"URL":"https:\/\/doi.org\/10.1099\/vir.0.81297-0","relation":{},"ISSN":["0022-1317","1465-2099"],"issn-type":[{"value":"0022-1317","type":"print"},{"value":"1465-2099","type":"electronic"}],"subject":[],"published":{"date-parts":[[2006,1,1]]}}}