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 language | English (US) |
|---|---|
| Pages (from-to) | 2573-2584 |
| Number of pages | 12 |
| Journal | Nucleic acids research |
| Volume | 46 |
| Issue number | 5 |
| DOIs | |
| State | Published - Mar 16 2018 |
Funding
National Institutes of Health (NIH) [R00HG006860 to S.A.]; New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health [1DP2GM110838 to J.B.L.]; Gates Foundation Grand Challenges [OPP 1068482 to J.R.T.]; K.E.W. was a Fleming Scholar in the Robert F. Smith School of Chemical and Biomolecular Engineering at Cornell University during part of this work. Funding for open access charge: Gates Foundation. National Institutes of Health (NIH) [R00HG006860 to S.A.]; New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health [1DP2GM110838 to J.B.L.]; Gates Foundation Grand Challenges [OPP 1068482 to J.R.T.]; K.E.W. was a Fleming Scholar in the Robert F. Smith School of Chemical and Biomolecular Engineering at Cornell University during part of this work. Funding for open access charge: Gates Foundation. Conflict of interest statement. None declared.
ASJC Scopus subject areas
- Genetics