RESUMO
Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.
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Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.
Assuntos
Antivirais , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Replicação Viral , Antivirais/farmacologia , Antivirais/química , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Animais , Humanos , Replicação Viral/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Ovinos , Farmacorresistência Viral , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Virais/genética , Pulmão/virologiaRESUMO
Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.
Assuntos
Dibenzocicloeptenos , Piridinas , Infecções por Vírus Respiratório Sincicial , Animais , Feminino , Camundongos , Reposicionamento de Medicamentos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/químicaRESUMO
Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.
Assuntos
Metapneumovirus , Criança , Humanos , Pré-Escolar , Metapneumovirus/genética , Nucleocapsídeo/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismoRESUMO
Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and antiviral drugs. RSV assembles and buds from the host cell plasma membrane by forming infectious viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of the matrix (M) protein with cell plasma membrane lipids forming a layer at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with cell membrane lipids, and with which one, to promote viral assembly. Here, we demonstrate that M proteins are able to cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as a dimer and upon M oligomerization, PS clustering is observed. In contrast, the presence of other negatively charged lipids like PI(4, 5)P2 does not enhance M binding beyond control zwitterionic lipids, while cholesterol negatively affects M interaction with membrane lipids. Moreover, we show that the initial binding of the RSV M protein with PS lipids is independent of the cytoplasmic tail of the fusion (F) glycoprotein (FCT). Here, we highlight that M binding on membranes occurs directly through PS lipids, this interaction is electrostatic in nature, and M oligomerization generates PS clusters.
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Vírus Sincicial Respiratório Humano , Humanos , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Montagem de Vírus , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Linhagem Celular TumoralRESUMO
Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.
Assuntos
RNA , Vírus Sincicial Respiratório Humano , Alcaloides de Veratrum , Corpos de InclusãoRESUMO
Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation.
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Vírus Sincicial Respiratório Humano , Criança , Idoso , Humanos , Vírus Sincicial Respiratório Humano/genética , Microscopia Crioeletrônica , Nucleocapsídeo/genética , RNA Viral/genética , Nucleoproteínas/genéticaRESUMO
Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.
Assuntos
Doenças dos Bovinos , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Bovino , Vírus Sincicial Respiratório Humano , Animais , Bovinos , Humanos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/veterinária , Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus Sincicial Respiratório Humano/metabolismo , Anticorpos AntiviraisRESUMO
The human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus. It is the major cause of severe acute lower respiratory tract infection in infants, the elderly population, and immunocompromised individuals. There is still no approved vaccine or antiviral treatment against RSV disease, but new monoclonal prophylactic antibodies are yet to be commercialized, and clinical trials are in progress. Hence, urgent efforts are needed to develop efficient therapeutic treatments. RSV RNA synthesis comprises viral transcription and replication that are catalyzed by the large protein (L) in coordination with the phosphoprotein polymerase cofactor (P), the nucleoprotein (N), and the M2-1 transcription factor. The replication/transcription is orchestrated by the L protein, which contains three conserved enzymatic domains: the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase or capping), and the methyltransferase (MTase) domain. These activities are essential for the RSV replicative cycle and are thus considered as attractive targets for the development of therapeutic agents. In this review, we summarize recent findings about RSV L domains structure that highlight how the enzymatic activities of RSV L domains are interconnected, discuss the most relevant and recent antivirals developments that target the replication/transcription complex, and conclude with a perspective on identified knowledge gaps that enable new research directions.
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Nucleoproteínas , Vírus Sincicial Respiratório Humano , Idoso , Lactente , Humanos , RNA Polimerase Dependente de RNA , Fatores de Transcrição , Anticorpos Monoclonais , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNARESUMO
BACKGROUND: The nucleoprotein (N protein) of respiratory syncytial virus (RSV) is a candidate antigen for new RSV vaccine development. The aim of the present study was to investigate the association between maternal antibody titers against the RSV N protein at birth and the newborns' risk of developing very severe lower respiratory tract infection (VS-LRTI). METHODS: In this single-center prospective cohort study, 578 infants born during the RSV epidemic season in France were included. Among these, 36 were hospitalized for RSV VS-LRTI. A generalized linear model was used to test the occurrence of a VS-LRTI in function of sex, mode of delivery, parity of the mother, type of pregnancy, date of birth in relation to the peak of the epidemic, and antibody titer against N protein. RESULTS: All cord blood samples had detectable antibodies against N protein. The mean titers were significantly lower in newborns with risk factors for RSV severe LRTI (preterm infants, birth before the peak epidemic, multiparous mother). There was no association between antibody titer against the N protein and a protection against VS-LRTI. CONCLUSIONS: Further studies are needed to support the hypothesis that transfer of maternal antibodies against the RSV N protein can provide a significant immune protection early in infancy and that N protein candidate vaccine may be a suitable target for maternal vaccine.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Lactente , Gravidez , Feminino , Recém-Nascido , Humanos , Recém-Nascido Prematuro , Estudos Prospectivos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Anticorpos AntiviraisRESUMO
Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in newborns, with all children being infected before the age of two. Reinfections are very common throughout life and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Recém-Nascido , Adulto , Criança , Idoso , Humanos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Anticorpos , Antivirais/farmacologiaRESUMO
The ongoing COVID-19 pandemic continues to pose a need for new and efficient therapeutic strategies. We explored antisense therapy using oligonucleotides targeting the severe acute respiratory syndrome coronavirus (SARS-CoV-2) genome. We predicted in silico four antisense oligonucleotides (ASO gapmers with 100% PTO linkages and LNA modifications at their 5' and 3'ends) targeting viral regions ORF1a, ORF1b, N and the 5'UTR of the SARS-CoV-2 genome. Efficiency of ASOs was tested by transfection in human ACE2-expressing HEK-293T cells and monkey VeroE6/TMPRSS2 cells infected with SARS-CoV-2. The ORF1b-targeting ASO was the most efficient, with a 71% reduction in the number of viral genome copies. N- and 5'UTR-targeting ASOs also significantly reduced viral replication by 55 and 63%, respectively, compared to non-related control ASO (ASO-C). Viral titration revealed a significant decrease in SARS-CoV-2 multiplication both in culture media and in cells. These results show that anti-ORF1b ASO can specifically reduce SARS-CoV-2 genome replication in vitro in two different cell infection models. The present study presents proof-of concept of antisense oligonucleotide technology as a promising therapeutic strategy for COVID-19.
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Human and bovine respiratory syncytial virus (HRSV and BRSV) are closely genetically related and cause respiratory disease in their respective host. Whereas HRSV vaccines are still under development, a multitude of BRSV vaccines are used to reduce clinical signs. To enable the design of vaccination protocols to entirely stop virus circulation, we aimed to investigate the duration, character and efficacy of the immune responses induced by natural infections. The systemic humoral immunity was monitored every two months during two years in 33 dairy cattle in different age cohorts following a natural BRSV outbreak, and again in selected individuals before and after a second outbreak, four years later. Local humoral and systemic cellular responses were also monitored, although less extensively. Based on clinical observations and economic losses linked to decreased milk production, the outbreaks were classified as moderate. Following the first outbreak, most but not all animals developed neutralising antibody responses, BRSV-specific IgG1, IgG2 and HRSV F- and HRSV N-reactive responses that lasted at least two years, and in some cases at least four years. In contrast, no systemic T cell responses were detected and only weak IgA responses were detected in some animals. Seronegative sentinels remained negative, inferring that no new infections occurred between the outbreaks. During the second outbreak, reinfections with clinical signs and virus shedding occurred, but the signs were milder, and the virus shedding was significantly lower than in naïve animals. Whereas the primary infection induced similar antibody titres against the prefusion and the post fusion form of the BRSV F protein, memory responses were significantly stronger against prefusion F. In conclusion, even if natural infections induce a long-lasting immunity, it would probably be necessary to boost memory responses between outbreaks, to stop the circulation of the virus and limit the potential role of previously infected adult cattle in the chain of BRSV transmission.
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Doenças dos Bovinos , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Bovino , Vírus Sincicial Respiratório Humano , Adulto , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , Bovinos , Doenças dos Bovinos/epidemiologia , Pré-Escolar , Humanos , Imunoglobulina A , Imunoglobulina G , Estudos Longitudinais , Infecções por Vírus Respiratório Sincicial/epidemiologiaRESUMO
Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.
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Nucleocapsídeo , Nucleoproteínas , RNA Viral , Vírus Sincicial Respiratório Humano , Empacotamento do Genoma Viral , Proteínas Estruturais Virais , Humanos , Nucleocapsídeo/química , Nucleocapsídeo/fisiologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/química , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismoRESUMO
Human RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via interactions with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the MED25 transactivator binding ACID domain on the one hand, and the C-terminal α3 helix of NS1, with an additional contribution of the globular domain of NS1, on the other hand. By NMR we show that the NS1 α3 sequence primarily binds to the MED25 ACID H2 face, similarly to the α-helical transactivation domains (TADs) of transcription regulators such as Herpex simplex VP16 and ATF6α, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 could compete with ATF6α TAD for binding to MED25. These findings point to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II.
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Complexo Mediador , Vírus Sincicial Respiratório Humano , Transativadores , Proteínas não Estruturais Virais , Cromatina/química , Humanos , Complexo Mediador/química , Ligação Proteica , Domínios Proteicos , RNA Polimerase II/metabolismo , Vírus Sincicial Respiratório Humano/genética , Transativadores/química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Animais , Humanos , Vírus Sincicial Respiratório Humano/fisiologia , Furões , Pulmão , Replicação Viral , Antivirais/farmacologia , TretinoínaRESUMO
The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.
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Nucleoproteínas , Vírus Sincicial Respiratório Humano , Vírus Sincicial Respiratório Humano/metabolismo , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Polarização de FluorescênciaRESUMO
Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (NNuc), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the NNuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and NNuc, which has not been demonstrated yet. Here, we finely characterized the binding P-NNuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc and that P binds to the N-terminal domain of N (NNTD), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-NNuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.