The capsid proteins of orthoflavivirus are interchangeable for West Nile virus assembly.

Orthoflavivirus virions consist of an envelope enclosing a nucleocapsid core. A critical step in orthoflavivirus assembly is the encapsidation of genomic RNA by the capsid protein to form the nucleocapsid. However, the molecular details of this process remain poorly understood, particularly whether capsid proteins bind genomic RNA through specific interactions. Here, we utilized West Nile virus (WNV) and dengue virus (DENV) as model systems to investigate heterologous genome RNA encapsidation. Employing a chimeric virus strategy, we demonstrated that the DENV-2 capsid protein can package WNV genomic RNA if two critical elements are preserved: cis-acting sequences essential for RNA synthesis and the cleavage site required for capsid maturation. Furthermore, by decoupling the WNV cis-acting elements from the capsid-coding sequence and replacing the WNV capsid protein with orthologous capsid proteins from other orthoflaviviruses, we provide direct virological evidence that orthoflavivirus capsid proteins package genomic RNAs in a non-sequence-specific manner. These findings advance our mechanistic understanding of orthoflavivirus genome packaging.IMPORTANCEThe encapsidation of genomic RNA by capsid proteins as the primary event in orthoflavivirus virion assembly is not well understood in current research; particularly, whether it involves specific packaging signals remains a question. The orthoflavivirus capsid protein plays a key role in both genome replication and viral particle assembly. Our comprehensive virological analyses reveal that orthoflavivirus capsid protein shows considerable interspecies packaging flexibility while critical replication and processing elements are preserved. These insights underscore a key aspect of orthoflavivirus biology: the conserved packaging function of capsid proteins across genus members is mediated by structural interactions, rather than sequence specificity.