Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by P _XD to N _TAIL Binding Strength

Abstract

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 (N _TAIL) 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 N _TAIL 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 N _TAIL 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 N _TAIL/XD K _D. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the N _TAIL to XD binding strength. Altogether, our data support a key role for XD binding to N _TAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Author Summary

Three proteins, the polymerase L, the phosphoprotein P and the nucleoprotein N, interplay to ensure transcription and replication of measles virus, a member of the Paramyxoviridae family. A regular array of nucleoprotein shields the viral genomic RNA. The resulting nucleocapsid constitutes the template of RNA synthesis used by the polymerase complex made of L and P, with the latter ensuring L anchoring onto the nucleocapsid. We herein report a correlation between the binding affinity of the C-terminal X domain of P (XD) and the intrinsically disordered C-terminal tail of N (N _TAIL), the ability to reinitiate the transcription at the intergenic regions and the accumulation rate of viral transcripts from recombinant viruses. We therefore propose that the N _TAIL/XD interaction contributes to maintaining the polymerase complex anchored onto the nucleocapsid while ending the upstream transcript and re-initiating the downstream transcript at every intergenic region. As such, the N _TAIL/XD interaction strength must be controlled so as to keep the viral transcription gradient within an optimal efficiency window. The conservation of this mode of interaction between the viral P and N proteins in many members of the Paramyxoviridae family reflects one of the major evolution constraints to which their polymerase machinery is subjected.