Structural and functional characterization of the coxsackievirus B3 CRE(2C): Role of CRE(2C) in negative- and positive-strand RNA synthesis

Mark J.M. van Ooij, Dorothee A. Vogt, Aniko Paul, Christian Castro, Judith Kuijpers, Frank J.M. van Kuppeveld, Craig Eugene Cameron, Eckard Wimmer, Raul Andino, Willem J.G. Melchers

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Abstract

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, R1NNNAAR2NNNNNNR3, 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 (A5G) 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.

Original languageEnglish (US)
Pages (from-to)103-113
Number of pages11
JournalJournal of General Virology
Volume87
Issue number1
DOIs
StatePublished - Jan 1 2006

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Enterovirus
RNA
Viral RNA
S 10
Rhinovirus
Poliovirus
Cell-Free System
Consensus Sequence
Site-Directed Mutagenesis
Cell Extracts
HeLa Cells
Adenosine
Genome
Mutation
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

van Ooij, M. J. M., Vogt, D. A., Paul, A., Castro, C., Kuijpers, J., van Kuppeveld, F. J. M., ... Melchers, W. J. G. (2006). Structural and functional characterization of the coxsackievirus B3 CRE(2C): Role of CRE(2C) in negative- and positive-strand RNA synthesis. Journal of General Virology, 87(1), 103-113. https://doi.org/10.1099/vir.0.81297-0
van Ooij, Mark J.M. ; Vogt, Dorothee A. ; Paul, Aniko ; Castro, Christian ; Kuijpers, Judith ; van Kuppeveld, Frank J.M. ; Cameron, Craig Eugene ; Wimmer, Eckard ; Andino, Raul ; Melchers, Willem J.G. / Structural and functional characterization of the coxsackievirus B3 CRE(2C) : Role of CRE(2C) in negative- and positive-strand RNA synthesis. In: Journal of General Virology. 2006 ; Vol. 87, No. 1. pp. 103-113.
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van Ooij, MJM, Vogt, DA, Paul, A, Castro, C, Kuijpers, J, van Kuppeveld, FJM, Cameron, CE, Wimmer, E, Andino, R & Melchers, WJG 2006, 'Structural and functional characterization of the coxsackievirus B3 CRE(2C): Role of CRE(2C) in negative- and positive-strand RNA synthesis', Journal of General Virology, vol. 87, no. 1, pp. 103-113. https://doi.org/10.1099/vir.0.81297-0

Structural and functional characterization of the coxsackievirus B3 CRE(2C) : Role of CRE(2C) in negative- and positive-strand RNA synthesis. / van Ooij, Mark J.M.; Vogt, Dorothee A.; Paul, Aniko; Castro, Christian; Kuijpers, Judith; van Kuppeveld, Frank J.M.; Cameron, Craig Eugene; Wimmer, Eckard; Andino, Raul; Melchers, Willem J.G.

In: Journal of General Virology, Vol. 87, No. 1, 01.01.2006, p. 103-113.

Research output: Contribution to journalArticle

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T1 - Structural and functional characterization of the coxsackievirus B3 CRE(2C)

T2 - Role of CRE(2C) in negative- and positive-strand RNA synthesis

AU - van Ooij, Mark J.M.

AU - Vogt, Dorothee A.

AU - Paul, Aniko

AU - Castro, Christian

AU - Kuijpers, Judith

AU - van Kuppeveld, Frank J.M.

AU - Cameron, Craig Eugene

AU - Wimmer, Eckard

AU - Andino, Raul

AU - Melchers, Willem J.G.

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N2 - 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, R1NNNAAR2NNNNNNR3, 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 (A5G) 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.

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