Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis.
A stem-loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R(1)NNNAAR(2)NNNNNNR(3), 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 in vitro 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(5)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.
van Ooij
Mark J M
MJM
Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Vogt
Dorothee A
DA
University of California, San Francisco, Mission Bay Genentech Hall, UCSF Department of Microbiology, 600 16th Street, PO Box 2280, San Francisco, CA 94143, USA.
Paul
Aniko
A
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Castro
Christian
C
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Kuijpers
Judith
J
Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
van Kuppeveld
Frank J M
FJM
Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Cameron
Craig E
CE
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Wimmer
Eckard
E
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Andino
Raul
R
University of California, San Francisco, Mission Bay Genentech Hall, UCSF Department of Microbiology, 600 16th Street, PO Box 2280, San Francisco, CA 94143, USA.
Melchers
Willem J G
WJG
Radboud University Medical Centre Nijmegen, Nijmegen Centre for Molecular Life Science, Department of Medical Microbiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016679:Genome, Viral; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D002352:Carrier Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral