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...

Dengue virus (DENV) is a mosquito-borne human pathogen that causes a serious public-health threat in tropical and subtropical regions of the world. Neither a vaccine to prevent nor an effective therapeutic agent to treat DENV infection is currently available. We established a stable cell line harboring a luciferase-reporting DENV subgenomic replicon to screen for inhibitors of DENV. A total of 14,400 small-molecule (MW < 500 Da) chemicals were evaluated for their ability to reduce luciferase reporter activity in cell lysates. One effective compound was identified from the screening. This compound was found to reduce virus production but did not block virus entry in virus-based assay. Mode-of-action analysis revealed that this inhibitor suppressed viral RNA replication but did not affect replicon translation. This compound potentially could be developed as an anti-DENV agent and might be useful for dissecting the molecular mechanism of DENV replication.