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Effect of mutations in the gene-end sequence on RSV transcription
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Edité par CCSD -
Expressing and multiplying – viral gene expression
Expressing and multiplying – viral gene expression.
The respiratory syncytial virus (RSV) genome is a single strand, negative sense RNA of about 15 kb that is packaged by the nucleoprotein (N) and maintained as a left-handed helical N-RNA ribonucleoprotein complex (RNP). This RNP is the template for two distinct activities: RNA replication and RNA transcription that generates 10 capped and poly-adenylated mRNAs. RNA transcription is carried out by the viral RNA-dependent RNA polymerase complex (RdRp), composed of the viral N, P, L and M2-1 proteins. RSV transcription proceeds through sequential stop-and-restart events, in which the RdRp recognizes gene start (GS) and gene end (GE) sequences that flank each gene and direct initiation and termination of transcription, respectively. By increasing the processivity of the RdRp, RSV transcription antiterminator protein M2-1 prevents premature transcription termination. M2-1 is an RNA binding protein that binds preferentially to GE and A-rich sequences present on RSV mRNAs. The exact mechanism of how M2-1 improves transcription and its relation with its RNA binding abilities remain to be clarified. In this work, the effect of GE sequence variation was analyzed by using a bicistronic RSV minigenome coding for Gaussia and Firefly luciferases. Relative expression of the Gaussia and Firefly luciferase reporters were compared between wild type or mutated GE sequences in the absence or in the presence of M2-1. The results highlighted the critical role of some nucleotides in the GE sequence for efficient transcription termination of the first gene and reinitiation at the second GS signal.
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