Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength
2016
Bloyet, Louis-Marie | Brunel, Joanna | Dosnon, Marion | Hamon, Véronique | Erales, Jenny | Gruet, Antoine | Lazert, Carine | Bignon, Christophe | Roche, Philippe | Longhi, Sonia | Gerlier, Denis | Immunobiologie des infections virales – Immunobiology of Viral Infections [CIRI] (CIRI-IbIV) ; Centre International de Recherche en Infectiologie (CIRI) ; École normale supérieure de Lyon (ENS de Lyon) ; Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL) ; Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon) ; Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL) ; Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | Centre International de Recherche en Infectiologie (CIRI) ; École normale supérieure de Lyon (ENS de Lyon) ; Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL) ; Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | Architecture et fonction des macromolécules biologiques (AFMB) ; Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS) | Centre de Recherche en Cancérologie de Marseille (CRCM) ; Aix Marseille Université (AMU)-Institut Paoli-Calmettes (IPC) ; Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | ANR-08-PCVI-0020,AFF-IDP,Determinants of specificity and affinity in partner recognition by intrinsically disordered proteins(2008) | ANR-11-INBS-0003,CRB-Anim,Réseau de Centres de Ressources Biologiques pour les animaux domestiques(2011)
International audience
显示更多 [+] 显示较少 [-]英语. Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.
显示更多 [+] 显示较少 [-]