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