Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein

Guryanov, Sergey; Liljeroos, Lassi; Kasaragod, P.; Kajander, Tommi; Butcher, Sarah J.

doi:10.1128/jvi.02865-15

Cited by 73 publications

(102 citation statements)

References 37 publications

Supporting

Mentioning

100

Contrasting

Order By: Relevance

“…If a hinge is present between the two N domains, it is likely that a more flexible linker is present in the N protein core. Based on the B factors, there is no such flexible linker between the two domains in either PIV5 or NiV N. Moreover, the crystal structure of a measles virus Ncore-P complex shows that the RNA-free monomeric N protein has a more collapsed conformation than that in the nucleocapsid (31). The conformation of the RNA-free N protein is therefore not related to how the sequestered RNA is unveiled during viral RNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…The P protein functions as a chaperone to keep the N protein monomeric before nucleocapsid assembly. The published structures showed that the N-terminal regions can bind at the sites that are involved in stabilizing the nucleocapsid, such as interactions for domain swapping or RNA binding (19,31). Once the monomeric N subunit is incorporated in the nucleocapsid, the P protein must be dissociated, and the interactions of the N subunits are established.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Severin

Terrell

Zengel

et al. 2016

J Virol

View full text Add to dashboard Cite

In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the ␣-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. Some pathogens appear to reemerge in spite of available vaccines, such as mumps virus and measles virus (3-5). Effective controls are needed to combat these pathogens. In order to develop more effective countermeasures, the mechanism of NSV replication should be better understood. One of the unique features in NSVs is that the genomic RNA is sequestered in the nucleocapsid (6). During transcription and replication, the viral RNA-dependent RNA polymerase (vRdRp) must be able to gain access to the sequestered genomic RNA in order to use it as the template. For Rhabdoviridae and Paramyxoviridae, the virus encodes a single nucleocapsid protein (N) that polymerizes as a linear capsid to encapsidate the genomic RNA (7). The viral polymerase complex consists of the large protein (L) and the phosphoprotein (P). IMPORTANCE Mumps virus (MuVThe structure of the nucleocapsid or a nucleocapsid-like particle has been solved for several members of Rhabdoviridae and Paramyxoviridae by X-ray crystallography or cryo-electron microscopy (cryo-EM) three-dimensional (3D) reconstruction (8-12). The common features among various structures are that the N protein has an N-terminal domain and C-terminal domain in its core, composed mostly of ␣-helices. When the N subunits assemble into a polymeric capsid, they are aligned in parallel in a linear fashion (13). There are extensive side-by-side interactions between the neighboring domains and domain swaps of extended loops and long termini. The genomic RNA is encapsidated in a cavity formed between the two core domains. Most of the RNA bases are stacked, some of which face the exterior and some the interior of the N protein core. The tight assembly of the nucleocapsid clearly suggests that vRdRp must open the N protein core in order to unveil the genomic RNA. How this action is carried out remains to be discovered. The interaction of the polymerase cofactor P with the nucleocapsid may provide some insights on this subject. The C-terminal domain of vesicular stomatitis virus (VSV) P protein binds between the extended loops in the C-terminal domains ...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Severin

Terrell

Zengel

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…Following synthesis, the N protein requires chaperoning by the P protein so as to be maintained in a soluble and monomeric form. 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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

“…The L polymerase processively synthesizes RNA transcripts only when associated with its P cofactor (1,(6)(7)(8)(9). Virus replication also requires the association of N and P to form soluble N 0 P complexes (10) that are used as encapsidation substrates (7). N 0 P possibly associates with the PϩL polymerase to form larger complexes (11,12).…”

mentioning

confidence: 99%

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

Bloyet

Welsch

Enchéry

et al. 2016

J Virol

View full text Add to dashboard Cite

Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCEViruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P؉L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses. Viruses with a nonsegmented negative-stranded RNA genome, or members of the order Mononegavirales, share a common and highly conserved genomic organization and replication machinery that are unique in the living world. Indeed, the L protein, or polymerase, which is endowed with all of the enzymatic activities required for the synthesis of RNA and the capping and polyadenylation of viral transcripts, cannot use naked viral genomic RNA in a processive way (1). Instead, L associates with the phosphoprotein (P) to dynamically anchor the polymerase complex to the nucleocapsid and/or to enable...

show abstract

Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein

Cited by 73 publications

References 37 publications

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

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

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

Contact Info

Product

Resources

About