In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.
Fiche publication
Date publication
mai 2018
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CATTENOZ Pierre
Tous les auteurs :
Smyth RP, Smith MR, Jousset AC, Despons L, Laumond G, Decoville T, Cattenoz P, Moog C, Jossinet F, Mougel M, Paillart JC, von Kleist M, Marquet R
Lien Pubmed
Résumé
Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.
Mots clés
5' Untranslated Regions, Genome, Viral, HEK293 Cells, HIV-1, genetics, Humans, Mutation, Nucleotide Motifs, Poly A, metabolism, RNA, Viral, biosynthesis, Regulatory Sequences, Ribonucleic Acid, Virus Assembly, Virus Replication
Référence
Nucleic Acids Res.. 2018 05 18;46(9):e57