Date Published: December 9, 2016
Publisher: Public Library of Science
Author(s): Louis-Marie Bloyet, Joanna Brunel, Marion Dosnon, Véronique Hamon, Jenny Erales, Antoine Gruet, Carine Lazert, Christophe Bignon, Philippe Roche, Sonia Longhi, Denis Gerlier, Roberto Cattaneo.
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.
Measles virus (MeV), a member of the Morbillivirus genus, belongs to the Paramyxoviridae family of the Mononegavirales order . These viruses possess a non-segmented RNA genome of negative polarity that is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. Not only does N protect viral RNA from degradation and/or formation of viral dsRNA, but it also renders the latter competent for transcription and replication. Indeed, the viral polymerase cannot processively transcribe nor replicate RNA unless the viral genome is encapsidated by the N protein within a helical nucleocapsid [2,3]. Transcription and replication are ensured by the RNA-dependent RNA polymerase complex made of the large protein (L) and the phosphoprotein (P), with P serving as an essential tethering factor between L and the nucleocapsid. The complex made of RNA and of the N, P and L proteins constitutes the replication machinery. In order to perform messenger RNA synthesis, the polymerase has not only to bind to the 3’ transcription promoter, but also to re-initiate the transcription of downstream genes upon crossing each intergenic region (IGR). Following polyadenylation, which serves as gene end (GE) signal, the polymerase proceeds over three nucleotides (3’-GAA-5’ or 3’-GCA-5’) without transcribing them and then restarts transcription upon recognition of a downstream gene start (GS) signal.
By combining in vitro biophysical and biochemical studies, in silico analyses (i.e. MD simulations) and in cellula polymerase functional investigations using recombinant viruses and dual-luciferase editing-dependent minigenome assays, we deciphered key molecular parameters that govern the NTAIL/XD interaction. Specifically, we uncovered a correlation between interaction strength and efficiency of transcription re-initiation at intergenic regions.