Self‐Assembly of Measles Virus Nucleocapsid‐like Particles: Kinetics and RNA Sequence Dependence

Milles, Sigrid; Jensen, Malene Ringkjøbing; Communie, Guillaume; Maurin, Damien; Schoehn, Guy; Ruigrok, Rob W.H.; Blackledge, Martin

doi:10.1002/anie.201602619

Cited by 42 publications

(65 citation statements)

References 30 publications

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“…Upon binding of two ssRNAs hexameric σNS assembles into octameric RNP complexes with defined stoichiometry. This mechanism of RNA-driven oligomerisation is distinct from that of other viral RNA-binding proteins that assemble into large, non-discrete, higher-order oligomers in the presence of RNA (59)(60)(61)(62). We propose that σNS hexamer assembles as a trimer of dimers (18), in dynamic equilibrium with a low octameric population (Figure 3).…”

Section: Rna-driven Oligomerisationmentioning

confidence: 85%

Stability of local secondary structure determines selectivity of viral RNA chaperones

Bravo

Borodavka

Barth

et al. 2018

Preprint

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To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between rotavirus genomic segment precursors. Despite their similar activities, NSP2 successfully promotes intersegment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerises into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

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Section: Rna-driven Oligomerisationmentioning

confidence: 85%

Stability of local secondary structure determines selectivity of viral RNA chaperones

Bravo

Borodavka

Barth

et al. 2018

Preprint

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“…The P N-terminal region (P NT ) binds to the neosynthesized N protein thereby simultaneously preventing its illegitimate self-assembly and yielding a soluble N 0 P complex the structure of which have been characterised for MeV [5] as well as for four other members of the Mononegavirales order [6,7,8,9]. N 0 P is used as the substrate for the encapsidation of the nascent genomic RNA chain during replication [10], (see also [4,11,12,13] for reviews on transcription and replication). In its assembled homopolymeric form or nucleocapsid, N also makes complexes with either isolated P or P bound to L, with all these interactions being essential for RNA synthesis by the viral polymerase [14,15,16].…”

Section: Introductionmentioning

confidence: 99%

“…it lacks highly populated secondary and tertiary structure under physiological conditions of pH and salinity in the absence of a partner (for a recent review on intrinsically disordered proteins see [20]). While N CORE contains all the regions necessary for self-assembly and RNA-binding [10,21,22] and a binding site for an α-MoRE located at the N terminus of the P protein, N TAIL is responsible for interaction with the C-terminal X domain (XD, aa 459–507) of P [11,18,21,23,24,25,26,27] ( Fig 1A ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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“…We do not know whether RNA sequence affects how N is transiently separated from the template RNA during the initiation of RNA synthesis. However, a fusion construct of MeV P and N proteins can act to assemble nucleocapsid-like particles in vitro, and this assembly was strongly sequence-dependent (Milles et al 2016). The displacement of the 3 ′ proximal N protomers from the template RNA during the initiation of RNA synthesis might similarly be sequence-sensitive, and the hPIV2 polypyrimidine run from 3 ′ U 4 to U 13 (Fig.…”

Section: Discussionmentioning

confidence: 99%

The control of paramyxovirus genome hexamer length and mRNA editing

Matsumoto

Ohta

Kolakofsky

et al. 2018

RNA

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The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NP) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NP is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the -acting mRNA editing sequence is maintained.

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Self‐Assembly of Measles Virus Nucleocapsid‐like Particles: Kinetics and RNA Sequence Dependence

Cited by 42 publications

References 30 publications

Stability of local secondary structure determines selectivity of viral RNA chaperones

Stability of local secondary structure determines selectivity of viral RNA chaperones

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

The control of paramyxovirus genome hexamer length and mRNA editing

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