Reversion to wildtype of a mutated and nonfunctional coxsackievirus B3CRE(2C)

Shane Smithee, Steven M Tracy, Nora M. Chapman

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] of enteroviruses (EV) facilitates the addition of two uridine residues (uridylylation) onto the virus-encoded protein VPg in order for it to serve as the RNA replication primer. We demonstrated that coxsackievirus B3 (CVB3) is replication competent in the absence of a native (uridylylating) CRE(2C) and also demonstrated that lack of a functional CRE(2C) led to generation of 5' terminal genomic deletions in the CVB3 CRE-. knock-. out (CVB3-CKO) population. We asked whether reversion of the mutated CRE(2C) occurred, thus permitting sustained replication, and when were 5' terminal deletions generated during replication. Virions were isolated from HeLa cells previously electroporated with infectious CVB3-CKO T7 transcribed RNA or from hearts and spleens of mice after transfection with CVB3-CKO RNA. Viral RNA was isolated in order to amplify the CRE(2C) coding region and the genomic 5' terminal sequences. Sequence analysis revealed reversion of the CVB3-CKO sequence to wildtype occurs by 8 days post-electroporation of HeLa cells and by 20 days post-transfection in mice. However, 5' terminal deletions evolve prior to these times. Reversion of the CRE(2C) mutations to wildtype despite loss of the genomic 5' termini is consistent with the hypothesis that an intact CRE(2C) is inherently vital to EV replication even when it is not enabling efficient positive strand initiation.

Original languageEnglish (US)
Pages (from-to)136-149
Number of pages14
JournalVirus Research
Volume220
DOIs
StatePublished - Jul 15 2016

Fingerprint

Enterovirus
HeLa Cells
Transfection
RNA
Electroporation
Uridine
Viral RNA
Virion
Open Reading Frames
Sequence Analysis
Spleen
Viruses
Mutation

Keywords

  • CRE(2C)
  • Coxsackievirus
  • Enterovirus
  • Reversion
  • Terminal deletion

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases
  • Cancer Research

Cite this

Reversion to wildtype of a mutated and nonfunctional coxsackievirus B3CRE(2C). / Smithee, Shane; Tracy, Steven M; Chapman, Nora M.

In: Virus Research, Vol. 220, 15.07.2016, p. 136-149.

Research output: Contribution to journalArticle

Smithee, Shane ; Tracy, Steven M ; Chapman, Nora M. / Reversion to wildtype of a mutated and nonfunctional coxsackievirus B3CRE(2C). In: Virus Research. 2016 ; Vol. 220. pp. 136-149.
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