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The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein.

ABSTRACT: Measles virus (MeV), like all viruses of the order Mononegavirales, utilizes a complex consisting of genomic RNA, nucleoprotein, the RNA-dependent RNA polymerase, and a polymerase cofactor, the phosphoprotein (P), for transcription and replication. We previously showed that a recombinant MeV that does not express another viral protein, C, has severe transcription and replication deficiencies, including a steeper transcription gradient than the parental virus and generation of defective interfering RNA. This virus is attenuated in vitro and in vivo However, how the C protein operates and whether it is a component of the replication complex remained unclear. Here, we show that C associates with the ribonucleocapsid and forms a complex that can be purified by immunoprecipitation or ultracentrifugation. In the presence of detergent, the C protein is retained on purified ribonucleocapsids less efficiently than the P protein and the polymerase. The C protein is recruited to the ribonucleocapsid through its interaction with the P protein, as shown by immunofluorescence microscopy of cells expressing different combinations of viral proteins and by split luciferase complementation assays. Forty amino-terminal C protein residues are dispensable for the interaction with P, and the carboxyl-terminal half of P is sufficient for the interaction with C. Thus, the C protein, rather than being an "accessory" protein as qualified in textbooks so far, is a ribonucleocapsid-associated protein that interacts with P, thereby increasing replication accuracy and processivity of the polymerase complex.IMPORTANCE Replication of negative-strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a cofactor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

SUBMITTER: Pfaller CK

PROVIDER: S-EPMC6997751 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein.

Pfaller Christian K CK Bloyet Louis-Marie LM Donohue Ryan C RC Huff Amanda L AL Bartemes William P WP Yousaf Iris I Urzua Erica E Clavière Mathieu M Zachary Marie M de Masson d'Autume Valentin V Carson Sandra S Schieferecke Adam J AJ Meyer Alyssa J AJ Gerlier Denis D Cattaneo Roberto R

Journal of virology 20200131 4

Measles virus (MeV), like all viruses of the order <i>Mononegavirales</i>, utilizes a complex consisting of genomic RNA, nucleoprotein, the RNA-dependent RNA polymerase, and a polymerase cofactor, the phosphoprotein (P), for transcription and replication. We previously showed that a recombinant MeV that does not express another viral protein, C, has severe transcription and replication deficiencies, including a steeper transcription gradient than the parental virus and generation of defective in ...[more]

PMID: 31748390

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Project description:UnlabelledThe genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.ImportanceMononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.

Project description:UnlabelledThe enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N(0)) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N(0) assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N(0)₁₋₄₀₈ fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N(0)-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N(0) binding peptide, P₁₋₄₈, to the C terminus of an N(0)₂₁₋₄₀₈ fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N(0)-P complex. We solved the X-ray structure of the resulting N(0)-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N(0)-P interface and explains how P chaperones N(0), preventing both self-assembly of N(0) and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization.ImportanceMeasles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.

Dataset Information

The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein.

Publications

The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein.

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