Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein

Renner, Max; Bertinelli, M.; Leyrat, C.; Paesen, Guido C.; Oliveira, Laura Freitas Saraiva de; Huiskonen, Juha T.; Grimes, Jonathan M.

doi:10.7554/elife.12627

Cited by 77 publications

(120 citation statements)

References 36 publications

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“…N mono leads to line broadening at the ␣ N1 site in P NDϩOD (Fig. 4, E and F), in agreement with previous results that showed that the N-terminal P 40 peptide was able to pull down N mono (24). NMR interaction experiments with N mono and P FL show that N mono is competent for both NC⅐P and N 0 ⅐P binding modes, via P NCBD and ␣ N1 , respectively (Fig.…”

Section: Extent Of Intrinsically Disordered Regions In Hrsv Phossupporting

confidence: 92%

“…4). Our definition of ␣ N1 matches with the second helix formed by hMPV P 1-28 in the X-ray structure of the N 0 ⅐P complex (40). The sequence of ␣ N1 is rather well conserved among Pneumoviridae (Fig.…”

Section: Discussionsupporting

confidence: 65%

“…4F and 5B), suggesting a difference in binding. We carried out complementary experiments by measuring spectra of 15 N-N NTD in the presence of P 40 . Line broadening was induced in different N NTD regions (Fig.…”

Section: Extent Of Intrinsically Disordered Regions In Hrsv Phosmentioning

confidence: 99%

“…The P 40 /N NTD interaction might therefore correspond to an intermediate state on the binding/folding pathway of ␣ N1 , whereby hydrophobic interactions play a role, as shown by the hydrophobic face in fully formed ␣ N1 (Fig. 6B), involved in N 0 binding (40). ␣ N1 appears to be a sticky helix that is able to mediate external (N/P) as well as internal interactions, the latter being favored by the tetrameric organization of P.…”

Section: Journal Of Biological Chemistry 2125mentioning

confidence: 99%

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New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

Pereira¹,

Cardone²,

Lassoued³

et al. 2017

Journal of Biological Chemistry

116

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Edited by Charles E. SamuelPhosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales. It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that Nand C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase. Human respiratory syncytial virus (hRSV),3 a member of the family Pneumoviridae (1) and order Mononegavirales (MNV), is the main viral cause of lower respiratory tract illness worldwide, and the main agent responsible for bronchiolitis and pneumonia in infants (2). All children have been infected by the age of two, requiring hospitalization in ϳ5% cases (3). Elderly and immunocompromised adults are also at increased risk. No efficient treatment is presently available for hRSV (4), and vaccination is challenging due to complex immunogenicity (5). The search for hRSV antiviral drugs directed toward specific viral functions is therefore still ongoing (6).The hRSV RNA-dependent RNA complex (RdRp) constitutes a virus-specific target with specific protein-protein interactions that have not all been investigated in detail (7). It uses the nonsegmented single-stranded negative sense RNA genome of hRSV as a template. In infected cells, the viral RdRp is found in specific inclusion bodies (8), which have been shown to be transcription and replication centers for other Mononegavirales, e.g. rabies (9) and vesicular stomatitis viruses (10). The apo RdRp complex is composed a minima of the large catalytic subunit (L) and its essential cofactor, the phosphoprotein (P) (11, 12). The P protein plays a central role in the RdRp by interacting with all main RdRp components. During transcription and replication it tethers the L protein to the nucleocapsid (NC), consisting of the genomic RNA packaged by the nucleoprotein (N), by direct interaction with N (13-16). hRSV P also binds to the transcription antitermination factor M2-1 (17-19). Phosphorylation of P has been proposed to regulate these interactions, although it is not essential for replication (20 -22). P also acts as a chaperone for neo-synthesized N by forming an N 0 ⅐P complex that preserves N in a monomeric and RNA-free state (23). We have shown previously that formation of hRSV NC⅐P and ...

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Section: Extent Of Intrinsically Disordered Regions In Hrsv Phossupporting

confidence: 92%

Section: Discussionsupporting

confidence: 65%

Section: Extent Of Intrinsically Disordered Regions In Hrsv Phosmentioning

confidence: 99%

Section: Journal Of Biological Chemistry 2125mentioning

confidence: 99%

See 2 more Smart Citations

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

Pereira¹,

Cardone²,

Lassoued³

et al. 2017

Journal of Biological Chemistry

116

View full text Add to dashboard Cite

show abstract

“…The genome is encapsidated by multiple copies of the nucleoprotein (N) to give rise to helical nucleocapsids (7). Additionally, copies of the phosphoprotein (P) interact with N and recruit the L protein, an RNA-dependent RNA polymerase, and the cofactor M2-1 to the viral nucleocapsids (8)(9)(10)(11). Estimates for Sendai virus, a paramyxovirus, show that a virion can contain up to 2,600 copies of N, 300 copies of P, and 50 copies of the L protein (12).…”

mentioning

confidence: 99%

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

Cifuentes-Muñoz

Branttie

Slaughter

et al. 2017

J Virol

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Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positivesense RNA (ϩRNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.KEYWORDS HMPV, pneumovirus, inclusion bodies, replication H uman metapneumovirus (HMPV) is a pathogen that affects all age groups worldwide, with an increased risk of severe disease being found in immunocompromised individuals, children under the age of 5 years, and the elderly (1-4). Together

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Negative‐strand RNA Viruses

Fearns

2021

Virology

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Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein

Cited by 77 publications

References 36 publications

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

Negative‐strand RNA Viruses

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