Structure of the Vesicular Stomatitis Virus N0-P Complex

Leyrat, C.; Yabukarski, Filip; Tarbouriech, Nicolas; Ribeiro, Euripedes de Almeida; Jensen, Malene Ringkjøbing; Blackledge, Martin; Ruigrok, Rob W.H.; Jamin, Marc

doi:10.1371/journal.ppat.1002248

Cited by 112 publications

(163 citation statements)

References 43 publications

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“…Such molecular flexibility also might be required to support the roles of P NTD as a chaperone for N 0 and as a binding site for L. The atomic structure of the N 0 -P1-60 complex reveals that P residues 6-40 form a molecular recognition element that engages N 0 , with amino acids 1-5 and 41-60 remaining flexible. Those flexible regions of P were suggested to act as "entropic bristles" that repel incoming RNA or N molecules or mask their binding interfaces (21). In the present study, the dispensability of the N 0 -binding molecular recognition element during transcription (P41-265) demonstrates the lack of effect of this region on polymerase function and is consistent with a primary role in the chaperoning N 0 during replication.…”

Section: Discussionsupporting

confidence: 78%

“…The role of P dimerization remains uncertain; however, it has been proposed to play a role in mediating the progression of L along the N-RNA template (20). The structure of N 0 lacking an N-terminal arm, in complex with a peptide corresponding to the first 60 residues of P, supports a model in which residues 6-35 of P block the polymerization of adjacent N molecules as well as access of RNA to the RNA-binding groove (21). Biophysical and bioinformatic analyses have indicated the presence of multiple intrinsically disordered regions (IDRs) separating the different structured domains of P (15).…”

mentioning

confidence: 94%

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Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Rahmeh¹,

Morin²,

Schenk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The RNA-dependent RNA polymerase (RdRP) of nonsegmented negative-sense RNA viruses consists of a large catalytic protein (L) and a phosphoprotein cofactor (P). During infection, the RdRP replicates and transcribes the viral genome, which resides inside an oligomer of nucleocapsid protein (N-RNA). The classical view of P as a cofactor for L assigns a primary role of P as a bridge mediating the access of L to the RNA template, whereby its N-terminal domain (P NTD ) binds L and its C-terminal domain (P CTD ) binds N-RNA. Recent biochemical and structural studies of a prototype nonsegmented negative-sense RNA virus, vesicular stomatitis virus, suggest a role for P beyond that of a mere physical link: P induces a structural rearrangement in L and stimulates polymerase processivity. In this study, we investigated the critical requirements within P mediating the functional interaction with L to form a fully functional RdRP. We analyzed the correlation between the impact of P on the conformation of L and its activity in RNA synthesis and the consequences of these events on RdRP function. We identified three separable elements of the P NTD that are required for inducing the conformational rearrangement of L, stimulating polymerase processivity, and mediating transcription of the N-RNA. The functional interplay between these elements provides insight into the role of P as a dynamic player in the RNA synthesis machine, influencing essential aspects of polymerase structure and function.large polymerase | replication and transcription | Mononegavirales | rhabdovirus T he functional unit necessary for transcription and replication of nonsegmented negative-sense (NNS) RNA viruses is a ribonucleoprotein (RNP) complex. The RNP complex comprises a genomic RNA encapsidated by a nucleocapsid protein oligomer (N-RNA), associated with the RNA-dependent RNA polymerase (RdRP) consisting of a complex of the large polymerase protein (L) and a phosphoprotein (P) (1-3). The template RNA is buried between the N-and C-terminal lobes of each N protomer; nevertheless, the overall integrity of the N-RNA structure is maintained during copying by the RdRP (4, 5). The L protein is the multifunctional catalytic core of the RNA synthesis machinery, harboring the RdRP as well as a capping enzyme and two methyltransferase activities that are required for mRNA synthesis (6-10). During RNA synthesis, L must gain access to the RNA, and its enzymatic activities must be regulated in accordance with a replicase or transcriptase mode of RNA synthesis. The access of L to the N-RNA is mediated by the noncatalytic cofactor P, which engages L and the N oligomer simultaneously (11,12).The functioning of an RNP as a highly regulated RNA synthesis machine requires an intricate, tight coordination of its individual components. The mechanisms that govern such functional coupling are largely unknown. Much of our understanding of the assembly, structure, and function of NNS RNA virus RNPs have come from studies of vesicular stomatitis virus (VSV). In part, this reflect...

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Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 94%

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Rahmeh¹,

Morin²,

Schenk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Yabukarski and colleagues (9) suggested that P 50 keeps the Nipah-N structure in the open conformation by rigidifying the helices in its CTD domain. However, VSV-N was stabilized in the closed conformation by its P 60 protein fragment (20), which also replaces its RNA in the VSV-N-binding pocket, implying that P does not induce open RNA-groove conformation. Our data suggests that N could exist naturally in an open conformation, regardless of the P fragment, due to charge repulsion between the positively charged roof and ceiling of the RNA binding pocket (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

Alayyoubi

Leser

Kors

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

135

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Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.nucleoprotein | nucleocapsid ring | atomic structure | paramyxovirus | ribonucleoprotein T he Paramyxoviridae family of nonsegmented negative-strand RNA viruses includes many serious pathogens of humans and animals including mumps virus, measles virus, parainfluenza viruses 1-5, Nipah virus, Hendra virus, and Newcastle disease virus, all of which are in the Paramyxovirinae subfamily, and respiratory syncytial virus (RSV) and human metapneumovirus, which are in the subfamily Pneumoviridae. The Paramyxoviridae belong in the order Mononegavirales of membrane enveloped negative-strand RNA viruses (NSV), an order that includes the Rhabdoviridae (rabies virus and vesicular stomatitis virus; VSV), Orthomyxoviridae (influenza virus) and Filoviridae (Ebola virus) (1). Parainfluenza virus 5 (PIV5) is a paramyxovirus that was initially isolated from rhesus monkey-kidney cell cultures (2) and although PIV5 is not known to cause human disease (3), it is used as a prototype in the study of paramyxoviruses.PIV5 has a nonsegmented single-stranded RNA genome of negative polarity of 15,246 nucleotides that encodes eight proteins (1): Three integral membrane proteins, fusion protein F and attachment protein hemagglutinin-neuraminidase (HN) and a small hydrophobic protein (SH). Inside the virion envelope lies a helical nucleocapsid core containing the R...

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“…Among Mononegavirales, the crystal structures of N 0 -P complexes have been resolved for one rhabdovirus (vesicular stomatitis virus [VSV]) and one paramyxovirus (Nipah virus), allowing to clarify the role of P as a chaperone protein (32,33). For VSV, the complex was reconstituted in vitro after purification of a recombinant N protein deleted of its 21 N-terminal residues, which was monomeric although still capable of interacting with RNA (32).…”

Section: Discussionmentioning

confidence: 99%

“…For VSV, the complex was reconstituted in vitro after purification of a recombinant N protein deleted of its 21 N-terminal residues, which was monomeric although still capable of interacting with RNA (32). A second step consisted of displacing RNA by a peptide, P[1-60], corresponding to residues 1 to 60 of the P protein.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

Galloux

Gabiane

Sourimant

et al. 2015

J Virol

113

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The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N 0 -P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N 0 monomer. We used this N mutant, designated N mono , as a substitute for N 0 in order to characterize the P regions involved in the N 0 -P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with N mono . We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N mono in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N 0 -P interaction could constitute a potential antiviral strategy. IMPORTANCERespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N 0 ) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus. T he human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (1). HRSV infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. It is also recognized as a significant cause of severe respiratory infections in the elderly. The virus belongs to the Mononegavirales order and constitutes the prototype virus of the Pneumovirus genus of the Paramyxoviridae family. As f...

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Structure of the Vesicular Stomatitis Virus N0-P Complex

Cited by 112 publications

References 43 publications

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

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