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.

呼吸道合胞病毒具有由病毒核蛋白 N 组成性包裹的负义单链 RNA 基因组，形成螺旋状核衣壳，是病毒聚合酶 L 进行病毒转录和复制的模板。磷蛋白 P，它是一种必需的 L 辅因子。基因组和反基因组衣壳化的先决条件是存在单体的、无 RNA 的、新合成的 N 蛋白，称为 N ⁰。N ⁰的稳定化取决于 P 的 N 末端残基与其表面的结合，这防止了 N 寡聚化。^{然而，从 N 0}过渡所涉及的机制-P 到核衣壳组装，因此在病毒基因组外壳化的特异性中，仍然未知。此外，虽然已证明与 RNA 复合的 P 和 N 之间的相互作用是造成这一过程的原因，但 N 寡聚化和 RNA 在发生病毒转录和复制的病毒工厂的形态发生中的具体作用尚未阐明。在这里，使用包含 N 和 P 的前 40 个 N 末端残基的嵌合蛋白，我们成功地纯化了一种重组的 N ^{0样蛋白，该蛋白能够}在体外进行 RNA 衣壳化. 我们的结果显示了 RNA 长度对于稳定外壳化的重要性，并揭示了 RNA 5' 末端的性质并不能解释外壳化的特异性。最后，我们证明了 RNA 衣壳化对于假病毒工厂的体外重组至关重要。总之，我们的研究结果提供了对呼吸道合胞病毒病毒基因组包裹特异性的深入了解。