51. An inhibitory motif on the 5'UTR of several rotavirus genome segments affects protein expression and reverse genetics strategies
- Author
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Catherine Eichwald, Francesca Arnoldi, Guido Papa, Oscar R. Burrone, Giuditta De Lorenzo, Marija Drikic, De Lorenzo, Giuditta, Drikic, Marija, Papa, Guido, Eichwald, Catherine, Burrone, Oscar R., Arnoldi, Francesca, and University of Zurich
- Subjects
RNA viruses ,0301 basic medicine ,Untranslated region ,Rotavirus ,Viral Diseases ,Five prime untranslated region ,Protein Expression ,Gene Expression ,lcsh:Medicine ,Pathology and Laboratory Medicine ,Biochemistry ,Genome ,UTR ,reverse genetics ,genome segments ,viral translation ,viral transcription ,viral mRNAs ,Transcription (biology) ,Reoviruses ,Medicine and Health Sciences ,lcsh:Science ,3' Untranslated Regions ,Genetics ,Multidisciplinary ,Messenger RNA ,reverse genetic ,Genomics ,DNA-Directed RNA Polymerases ,Complementary DNA ,Nucleic acids ,Infectious Diseases ,Medical Microbiology ,Viral Pathogens ,Viruses ,Pathogens ,Sequence Analysis ,Research Article ,10244 Institute of Virology ,Gene Expression Regulation, Viral ,DNA, Complementary ,Forms of DNA ,Molecular Sequence Data ,Genome, Viral ,1100 General Agricultural and Biological Sciences ,Biology ,Research and Analysis Methods ,Microbiology ,Viral Proteins ,03 medical and health sciences ,Sequence Motif Analysis ,1300 General Biochemistry, Genetics and Molecular Biology ,Gene Expression and Vector Techniques ,Nucleotide Motifs ,Molecular Biology Techniques ,Sequencing Techniques ,Gene Prediction ,Molecular Biology ,Microbial Pathogens ,Rotavirus Infection ,genome segment ,RNA, Double-Stranded ,Molecular Biology Assays and Analysis Techniques ,1000 Multidisciplinary ,Biology and life sciences ,Three prime untranslated region ,Alternative splicing ,lcsh:R ,Organisms ,Computational Biology ,DNA ,Genome Analysis ,Rotaviru ,Molecular biology ,Reverse genetics ,Alternative Splicing ,Open reading frame ,030104 developmental biology ,RNA ,570 Life sciences ,biology ,Protein Translation ,lcsh:Q ,5' Untranslated Regions - 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.
- Published
- 2016