Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

PLoS Pathogens

Denis Gerlier¹  Louis-Marie Bloyet²  Antoine Gruet²  Sonia Longhi³  Jenny Erales³  Marion Dosnon⁴  Christophe Bignon⁴  Joanna Brunel⁵  Carine Lazert⁵  Véronique Hamon⁶  Philippe Roche⁶ 

[1]Aix-Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, Marseille, France

[2]CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France

[3]CNRS, UMR5308, Lyon, France

[4]Ecole Normale Supérieure de Lyon, Lyon, France

[5]INSERM, U1111, Lyon, France

[6]Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France

关键词: Polymerases;    Nucleocapsids;    Luciferase;    DNA transcription;    Viral gene expression;    Hydrogen bonding;    RNA synthesis;    Messenger RNA;   

DOI  :  10.1371/journal.ppat.1006058

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY