The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal ␣2 and ␣4 helices of each monomer. Pairing of ␣2-␣2= creates a continuous hydrophobic surface, while the ␣4-␣4= helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the ␣4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the ␣4-␣4= helix pair conformation is critical in preserving the overall ␣-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the ␣4-␣4= helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. IMPORTANCEStructure-based mutagenesis study of the dengue virus core protein (CP) reveals that the ␣4-␣4= helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected. In vitro inefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation for in vivo virus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by ␣4-␣4= helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the ␣4-␣4= helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production. V iruses of the Flaviviridae family are enveloped and have a monopartite, linear, and positive-polarity single-stranded RNA genome. A member of this family in the Flavivirus genus, Dengue virus (DENV), is the most important human arthropodborne viral pathogen, putting 3 billion people at risk and resulting in 50 million to 100 million infections and around 22,000 deat...

Edited by Michael IbbaKeywords: DENV Core protein NS5 NS3 helicase RNA chaperone Strand annealing RNA binding protein a b s t r a c tIn this study we showed that the dengue virus (DENV) core protein forms a dimer with an a-helixrich structure, binds RNA and facilitates the strand annealing process. To assess the RNA chaperone activity of this core protein and other dengue viral RNA-interacting proteins, such as NS3 helicase and NS5 proteins, we engineered cis-and trans-cleavage hammerhead ribozyme constructs carrying DENV genomic RNA elements. Our results indicate that DENV core protein facilitates typical hammerhead structure formation by acting as an RNA chaperone and DENV NS5 has a weak RNA chaperone activity, while DENV NS3 helicase failed to refold RNA with a complex secondary structure.