Solution and Crystallographic Structures of the Central Region of the Phosphoprotein from Human Metapneumovirus

Leyrat, C.; Renner, Max; Harlos, K.; Grimes, Jonathan M.

doi:10.1371/journal.pone.0080371

Cited by 38 publications

(55 citation statements)

References 75 publications

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“…The presence of transient C-terminal helices was also proposed for hMPV, based on small angle X-ray scattering data (45), showing that they constitute another hallmark of Pneumoviridae. But whereas ␣ C1 is partly conserved, ␣ C2 appears to be specific of the Orthopneumovirus genus (Fig.…”

Section: Discussionmentioning

confidence: 91%

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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: Discussionmentioning

confidence: 91%

“…6A). Its structure was solved by X-ray diffraction for hMPV P (45). It displays a coiled-coil helical conformation for a region equivalent to the hRSV fragment Y*, indicating that a short OD is specific of this family.…”

Section: Discussionmentioning

confidence: 99%

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

“…In this context, we hypothesize that the HMPV P protein could have a crucial role, as it has been shown to be closely associated with actin and has a dual function in viral replication and assembly/spread (34,50). The HMPV P protein has been reported to homotetramerize, to have regions of intrinsic disorder, and to interact with the N protein (8,51,52). Future work will further characterize other cellular components with which the P protein interacts and also determine whether membrane-associated proteins are recruited to HMPV IBs, as is the case for Marburg virus (53).…”

Section: Discussionmentioning

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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“…The self-association of P is required for transcriptional activity, and the binding site for SeV L was found to neighbor the oligomerization region (5,(7)(8)(9). Tetrameric P structures have also been observed for other paramyxoviruses, with crystallization of the P oligomerization domains being found for measles virus, human metapneumovirus, and mumps virus (10)(11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 94%

Oligomerization of Mumps Virus Phosphoprotein

Pickar

Elson

Yang

et al. 2015

J Virol

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The mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (P N ), the oligomerization domain (P O ), and the C-terminal domain (P C ). While P N is known to relax the NP-bound RNA genome, the roles of P O and P C are not clear. In this study, we investigated the roles of P O and P C in viral RNA synthesis using mutational analysis and a minigenome system. We found that P N and P C functions can be trans-complemented. However, this complementation requires P O , indicating that P O is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (P N to P N and P C to P C ). Furthermore, we found that residues R231, K238, K253, and K260 in P O are critical for P's functions. We identified P C to be the domain that interacts with L. These results provide structure-function insights into the role of MuV P. IMPORTANCEMuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented in trans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals. Mumps virus (MuV) is a human pathogen of the Rubulavirus genus of the Paramyxoviridae family that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis (1). The nonsegmented, negativestranded RNA genome of MuV contains 15,384 nucleotides and encodes nine viral proteins (1). The viral RNA is encapsidated by the nucleoprotein (NP), and this helical nucleocapsid (RNP) functions as the template for viral RNA synthesis. Together, the large protein (L) and the phosphoprotein (P) make up the viral RNA-dependent RNA polymerase (vRdRp) (2). The enzymatic activities of the L protein involve the initiation, elongation, and termination of RNA synthesis, as well as mRNA capping (3). While P is not known to have intrinsic enzymatic activity, P is an essential cofactor of the polymerase. P oligomerizes by itself and forms complexes with L, NP, and RNP. It is thought that P docks the vRdRP to RNP (4).The P proteins of paramyxoviruses are modular and consist of N-terminal (P N ), oligomerization (P O ), and C-terminal (P C ) domains with flexible linkers between adjoining domains. The selfassociation of P is observed throughout negative-stranded RNA viruses (NSVs). The oligomerization domain of Sendai virus (SeV) P was the first to be crystallized, and those studies revealed a parallel coiled-coil tetramer (5, 6). The self-association of P is required for transcriptional activity, and the binding site for SeV L was found to neighbor the oligomerizat...

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Solution and Crystallographic Structures of the Central Region of the Phosphoprotein from Human Metapneumovirus

Cited by 38 publications

References 75 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

Oligomerization of Mumps Virus Phosphoprotein

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