An inhibitory motif on the 5'UTR of several rotavirus genome segments affects protein expression and reverse genetics strategies

De Lorenzo, G. , Drikic, M., Papa, G., Eichwald, C., Burrone, O. R. and Arnoldi, F. (2016) An inhibitory motif on the 5'UTR of several rotavirus genome segments affects protein expression and reverse genetics strategies. PLoS ONE, 11(11), e0166719. (doi: 10.1371/journal.pone.0166719) (PMID:27846320) (PMCID:PMC5112996)

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Abstract

Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies.

Item Type:Articles
Additional Information:FA was partially supported by a FIRB-Futuro in Ricerca grant (RBFR13209E, http://futuroinricerca.miur.it) funded by the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), Italy.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:De Lorenzo, Dr Giuditta
Authors: De Lorenzo, G., Drikic, M., Papa, G., Eichwald, C., Burrone, O. R., and Arnoldi, F.
College/School:College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN:1932-6203
ISSN (Online):1932-6203
Copyright Holders:Copyright © 2016 De Lorenzo et al.
First Published:First published in PLoS ONE 11(11): e0166719
Publisher Policy:Reproduced under a Creative Commons License

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