Etude structurale et fonctionnelle de la nucléocapside du virus respiratoire syncytial

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in young children. It is also responsible for severe respiratory infections in elderly or immunocompromised people. There is no vaccine or specific treatment for RSV.RSV is an enveloped virus with a single-stranded negative-sense RNA genome containing 10 genes coding for 11 proteins. The viral genome is constantly encapsidated by multiple copies of the viral nucleoprotein N, constituting a helical N-RNA complex, called the nucleocapsid. During the viral cycle, the nucleocapsid is used as a template by the viral polymerase L to (i) transcribe viral messenger RNAs, and (ii) replicate genomes and antigenomes. The activities of the polymerase L require interaction with its main cofactor, the phosphoprotein P, as well as the transcription anti-terminator protein M2-1. P can be seen as the hub of the virus multiplication, able to interact with the polymerase L, the nucleocapsid, M2-1, but also with the neosynthesised N, allowing its maintenance in a monomeric and RNA-free form, called N0. Viral transcription and replication steps take place in viral factories, in the cytoplasm of the infected cells. Viral factories are membrane-less organelles formed by liquid-liquid phase separation, whose morphogenesis depends on the expression of N and P. N, P, and their interactions, therefore, play a central role not only in the function of the polymerase L but also in the formation of viral factories. However, the mechanisms regulating the specificity of viral genomic and antigenomic RNA encapsidation, dependent on the N0-P to N-RNA transition, remain poorly characterised. Furthermore, high-resolution structural information is limited to the crystallographic structure of RSV N-RNA rings. In this context, my thesis work aimed at obtaining functional and structural data on the RSV nucleocapsid, following two main axes: (i) the study of the role of RNA in the encapsidation by N and (ii) the determination of the structure of the RSV nucleocapsid by cryo-electron microscopy. Starting from the ability to purify a recombinant monomeric and RNA-free N0 expressed in E. coli, I first studied the propensity of this protein to encapsidate different synthetic RNAs in vitro. The data obtained showed that encapsidation can be triggered in the presence of a 7 nucleotides-long RNA, but that 11 nucleotides are required to form stable N-RNA complexes in vitro. Secondly, the nature of the 5' end of the RNAs does not explain the specificity of encapsidation. Finally, we have shown that RNA encapsidation by the N protein is essential for the reconstitution of pseudo-viral factories in vitro. In parallel, the purification of nucleocapsids expressed in insect cells allowed us to determine the cryo-electron microscopy structures of several N-RNA assemblies (helical nucleocapsid, double-headed helical nucleocapsid, ring-capped helical nucleocapsid, and N-RNA double ring). Specifically, our data revealed that the RSV nucleocapsid exhibits a non-canonical symmetry, with an asymmetric unit composed of 16 N protomers. This particular assembly leads to a variation in the RNA accessibility along the helix. Finally, we have shown that this non-canonical symmetry depends on the C-terminal arm of the N protein since its truncation leads to the formation of a helical nucleocapsid of canonical symmetry.Le virus respiratoire syncitial (VRS) est la première cause de bronchiolites et de pneumonies chez les jeunes enfants. Il est également responsable d'infections respiratoires sévères chez les personnes âgées ou immunodéprimées. Il n'existe pas de vaccin ni de traitement spécifique contre le VRS. Le VRS est un virus enveloppé dont le génome est un ARN simple-brin de polarité négative contenant 10 gènes codant pour 11 protéines virales. Le génome viral est constamment encapsidé par de multiples copies de la nucléoprotéine virale N, constituant un complexe N-ARN en forme d'hélice, appelé nucléocapside. Au cours du cycle viral, la nucléocapside est utilisée comme matrice par la polymérase virale L pour (i) transcrire les ARN messagers viraux, et (ii) répliquer génomes et antigénomes. Les activités de la polymérase L dépendent de ses cofacteurs viraux, la phosphoprotéine P et la protéine M2-1, anti-terminateur de la transcription. La protéine P peut être vue comme le chef d'orchestre de la multiplication du virus, capable d'interagir avec la polymérase L, la nucléocapside, la protéine M2-1, mais également avec la protéine N néosynthétisée, permettant ainsi son maintien sous forme monomérique et non liée à l'ARN, nommée N0. Les étapes de transcription et de réplication se déroulent au sein d'usines virales, dans le cytoplasme de la cellule infectée. Les usines virales sont des organelles dépourvues de membranes, formées par séparation de phase liquide, dont la morphogénèse dépend de l'expression des protéines N et P. Les protéines N et P et leurs interactions jouent par conséquent un rôle central non seulement pour le fonctionnement de la polymérase mais également dans la formation des usines virales. Toutefois, les mécanismes régulant la spécificité d'encapsidation des ARN génomique et antigénomique viraux, dépendant de la transition N0-P vers N-ARN, restent mal caractérisés. De plus, les informations structurales de haute résolution sont limitées à la structure cristallographique d'anneaux N-ARN du VRS. Dans ce contexte, mes travaux de thèse visaient à obtenir des données fonctionnelles et structurales sur la nucléocapside du VRS, suivant deux axes principaux : (i) l'étude du rôle de l'ARN dans l'encapsidation par la protéine N et (ii) la détermination de la structure de la nucléocapside du VRS par cryo-microscopie électronique. Partant de la capacité à purifier une protéine N recombinante exprimée chez E. coli sous forme monomérique et non liée à l'ARN, j'ai dans un premier temps étudié la capacité de cette protéine à encapsider différents ARN synthétiques in vitro. Les données obtenues ont permis de montrer que l'encapsidation peut se déclencher en présence d'ARNs de 7 nucléotides, mais que 11 nucléotides sont nécessaires pour former des complexes N-ARN stables in vitro. Cependant, la nature de l'extrémité 5' des ARNs ne permet pas d'expliquer la spécificité d'encapsidation. Enfin, ces travaux ont permis de montrer que l'encapsidation de l'ARN par la protéine N est indispensable pour la reconstitution de pseudo-usines virales in vitro. En parallèle, la purification de nucléocapsides exprimées en cellules d'insectes nous a permis de déterminer la structure par cryo-microscopie électronique de plusieurs assemblages N-ARN (nucléocapside hélicoïdale, nucléocapside hélicoïdale à deux têtes, nucléocapside hélicoïdale coiffée d'un anneau, et double-anneaux N-ARN). Plus spécifiquement, nos données ont permis de révéler que la nucléocapside du VRS présente une symétrie non-canonique, avec un assemblage en unité asymétrique composée de 16 protomères de N. Cet assemblage particulier conduit à une variation dans l'accessibilité de l'ARN le long de l'hélice. Enfin, nous avons montré que cette symétrie non-canonique dépendait du bras C-terminal de la protéine N, puisque son raccourcissement entraine la formation d'une nucléocapside hélicoïdale de symétrie canonique

Interactions between the nucleoprotein and the phosphoprotein of pneumoviruses: structural insight for rational design of antivirals

International audiencePneumoviruses include pathogenic human and animal viruses, the most known and studied being the human respiratory syncytial virus (hRSV) and the metapneumovirus (hMPV), which are the major cause of severe acute respiratory tract illness in young children worldwide, and main pathogens infecting elderly and immune-compromised people. The transcription and replication of these viruses take place in specific cytoplasmic inclusions called inclusion bodies (IBs). These activities depend on viral polymerase L, associated with its cofactor phosphoprotein P, for the recognition of the viral RNA genome encapsidated by the nucleoprotein N, forming the nucleocapsid (NC). The polymerase activities rely on diverse transient protein-protein interactions orchestrated by P playing the hub role. Among these interactions, P interacts with the NC to recruit L to the genome. The P protein also plays the role of chaperone to maintain the neosynthesized N monomeric and RNA-free (called N0) before specific encapsidation of the viral genome and antigenome. This review aims at giving an overview of recent structural information obtained for hRSV and hMPV P, N, and more specifically for P-NC and N0-P complexes that pave the way for the rational design of new antivirals against those viruses

Structural landscape of the respiratory syncytial virus nucleocapsids

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

In vitro study of RNA encapsidation by the nucleoprotein of human Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) 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, neosynthesised N protein, named N 0. Stabilisation of N 0 depends on the binding of the N-terminal residues of P to its surface, that prevents N oligomerisation. However, the mechanism involved in the transition from N 0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, although the interaction between P and N complexed to RNA has been shown to be responsible for the morphogenesis of viral factories, where viral transcription and replication occur, the specific role of N oligomerisation and RNA in this process has not been elucidated. In the present study, using a chimeric protein between N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N 0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed differences in encapsidation depending on the nature of the 5' end, without any specificity for RNA sequence. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories

In vitro study of RNA encapsidation by the nucleoprotein of human Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) 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, neosynthesised N protein, named N 0. Stabilisation of N 0 depends on the binding of the N-terminal residues of P to its surface, that prevents N oligomerisation. However, the mechanism involved in the transition from N 0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, although the interaction between P and N complexed to RNA has been shown to be responsible for the morphogenesis of viral factories, where viral transcription and replication occur, the specific role of N oligomerisation and RNA in this process has not been elucidated. In the present study, using a chimeric protein between N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N 0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed differences in encapsidation depending on the nature of the 5' end, without any specificity for RNA sequence. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories

In vitro study of RNA encapsidation by the nucleoprotein of human Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) 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, neosynthesised N protein, named N 0. Stabilisation of N 0 depends on the binding of the N-terminal residues of P to its surface, that prevents N oligomerisation. However, the mechanism involved in the transition from N 0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, although the interaction between P and N complexed to RNA has been shown to be responsible for the morphogenesis of viral factories, where viral transcription and replication occur, the specific role of N oligomerisation and RNA in this process has not been elucidated. In the present study, using a chimeric protein between N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N 0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed differences in encapsidation depending on the nature of the 5' end, without any specificity for RNA sequence. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories

Depletion of TAX1BP1 amplifies innate immune responses during respiratory syncytial virus infection

International audienceRespiratory syncytial virus (RSV) is the main cause of acute respiratory infections in young children, and also has a major impact on the elderly and immunocompromised people. In the absence of a vaccine or efficient treatment, a better understanding of RSV interactions with the host antiviral response during infection is needed. Previous studies revealed that cytoplasmic inclusion bodies (IBs) where viral replication and transcription occur could play a major role in the control of innate immunity during infection by recruiting cellular proteins involved in the host antiviral response. We recently showed that the morphogenesis of IBs relies on a liquid-liquid phase separation mechanism depending on the interaction between viral nucleoprotein (N) and phosphoprotein (P). These scaffold proteins are expected to play a central role in the recruitment of cellular proteins to IBs. Here, we performed a yeast two-hybrid screen using RSV N protein as a bait, and identified the cellular protein TAX1BP1 as a potential partner of this viral protein. This interaction was validated by pulldown and immunoprecipitation assays. We showed that TAX1BP1 suppression has only a limited impact on RSV infection in cell cultures. However, RSV replication is decreased in TAX1BP1-deficient mice (TAX1BP1 KO ), whereas the production of inflammatory and antiviral cytokines is enhanced. In vitro infection of wild-type or TAX1BP1 KO alveolar macrophages confirmed that the innate immune response to RSV infection is enhanced in the absence of TAX1BP1. Altogether, our results suggest that RSV could hijack TAX1BP1 to restrain the host immune response during infection. Importance Respiratory syncytial virus (RSV), which is the leading cause of lower respiratory tract illness in infants, still remains a medical problem in the absence of vaccine or efficient treatment. This virus is also recognized as a main pathogen in the elderly and immunocompromised people, and the occurrence of co-infections (with other respiratory viruses and bacteria) amplifies the risks of developing respiratory distress. In this context, a better understanding of the pathogenesis associated to viral respiratory infections, which depends on both viral replication and the host immune response, is needed. The present study reveals that the cellular protein TAX1BP1, which interacts with the RSV nucleoprotein N, participates in the control of the innate immune response during RSV infection, suggesting that N-TAX1BP1 interaction represents a new target for the development of antivirals