Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

Kyle E. Watters, Krishna Choudhary, Sharon Aviran, Julius B. Lucks, Keith L. Perry, Jeremy R. Thompson

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities fromoverlapping reverse transcriptase priming sites.

Original languageEnglish (US)
Pages (from-to)2573-2584
Number of pages12
JournalNucleic Acids Research
Volume46
Issue number5
DOIs
StatePublished - Jan 1 2018

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Acylation
RNA Viruses
Plant Cells
Hydroxyl Radical
Open Reading Frames
Cucumovirus
RNA
Pressure
RNA-Directed DNA Polymerase
Capsid Proteins
3' Untranslated Regions
Virus Diseases
Tobacco
Genome
Viruses
Mutation
Infection
In Vitro Techniques
Silent Mutation

ASJC Scopus subject areas

  • Genetics

Cite this

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements. / Watters, Kyle E.; Choudhary, Krishna; Aviran, Sharon; Lucks, Julius B.; Perry, Keith L.; Thompson, Jeremy R.

In: Nucleic Acids Research, Vol. 46, No. 5, 01.01.2018, p. 2573-2584.

Research output: Contribution to journalArticle

Watters, Kyle E. ; Choudhary, Krishna ; Aviran, Sharon ; Lucks, Julius B. ; Perry, Keith L. ; Thompson, Jeremy R. / Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements. In: Nucleic Acids Research. 2018 ; Vol. 46, No. 5. pp. 2573-2584.
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