Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P and P–L interactions using a minigenome system

113

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...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Galloux

Gabiane

Sourimant

et al. 2015

113

“…The RSV P protein interacts with N (14) and, as shown for bovine RSV (bRSV), with the L protein (23). In both RSVinfected and cotransfected cells, M2-1 has been observed in inclusion bodies, but only in the presence of N and P proteins (14).…”

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confidence: 99%

Interaction between Human Respiratory Syncytial Virus (RSV) M2-1 and P Proteins Is Required for Reconstitution of M2-1-Dependent RSV Minigenome Activity

Mason

Aberg

Lawetz

et al. 2003

We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.Respiratory syncytial virus (RSV) is a nonsegmented negative-strand virus of the family Paramyxoviridae in the genus Pneumovirus (4). The viral genome is composed of 10 genes that are transcribed by the viral RNA polymerase complex (4). The activity of the RSV RNA-dependent RNA polymerase minimally requires the viral proteins N, P, and L, as well as signals in the viral RNA genome (17, 33). For RNA replication, the sequences in the leader and trailer regions function as the promoters for genome and antigenome replication, respectively (11). For mRNA synthesis, the gene-start and gene-end (GE) sequences which frame each RSV gene, in addition to the leader region, are essential (25). Viral transcription occurs sequentially along the genome in a stop-start manner characterized by attenuation at each GE sequence (4). An additional RSV protein, M2-1, is known to be important for transcription by the RSV polymerase, particularly on longer genes. M2-1 functions as an elongation factor, allowing the complete synthesis of RSV mRNAs (6) and as an antitermination factor to permit transit through GE and intergenic regions which allows the RSV polymerase access to promoter-distal genes (10,18,19).The RSV P protein interacts with N (14) and, as shown for bovine RSV (bRSV), with the L protein (23). In both RSVinfected and cotransfected cells, M2-1 has been observed in inclusion bodies, but only in the presence of N and P proteins (14). This colocalization of M2-1 with N and P would be consistent with its function in transcription as part of the ribonucleoprotein (RNP) complex. In that study (14), an interaction between M2-1 and N was observed by coimmunoprecipitation (co-IP). Others have also observed an interaction between M2-1 and N (20), although this interaction is believed to be mediated by RNA (3). Yeast two-hybrid analysis of pairwise combinations of these three RSV proteins did not reveal interacting partners for M2-1 (21).In an effort to identify protein-protein interactions between components of the RSV RNP, we investigated potential interactions between human RS...

“…Efficient and specific recognition of the RNP template by the RdRp is critical for viral RNA synthesis. It is mediated by P, a multifunctional protein capable of interacting with multiple partners: L (6), N (7), and M2-1 (8), but also cellular proteins (9). P positions the RdRp complex on the RNP and is likely involved in translocation along the RNP (10).…”

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confidence: 99%

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus

Ouizougun-Oubari

Pereira

Tarus

et al. 2015

Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCERespiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals. H uman respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in infants worldwide (1).No RSV vaccine is presently available, and the development of pediatric vaccines is particularly challenging. Currently, antiviral therapy is limited to palivizumab, a humanized mouse monoclonal antibody that targets the RSV fusion protein and is licensed for prophylactic use, and ribavirin, which has been used to treat severe infections despite its toxicity, its teratogenicity, and the limited evidence...