Increased capsid oligomerization is deleterious to dengue virus particle production.

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

Generation and characterization of luciferase-secreting, single-round infectious DENV-2 reporter for functional antibody assays.

Flavivirus reporters provide a robust tool for viral pathogenesis studies, anti-viral drug screening, disease diagnosis and functional antibody assays. In this study, we generated a luciferase-secreting, single-round reporter virus by replacing the capsid coding region in a DENV-2 genome with the secretory form of Lucia luciferase gene to produce infectious viral particles in a stable capsid-expressing mosquito cell line. Replication of the reporter virus in trans-complementing mosquito cells was sustained for up to two weeks. There were strong correlations between the extracellular luciferase activity and infectious reporter virus inocula upon infection of mosquito and mammalian cell lines with graded quantities of the reporter virus. A set of anti-E and anti-prM monoclonal antibodies affected the infectivity of reporter virus with similar dose-effect relationships as the parent virus. This simplified version of DENV-2 reporter provides a rapid and reliable method for the detection of neutralizing and infection-enhancing antibodies against dengue virus.

Enhanced production of infectious particles by adaptive modulation of C-prM processing and C-C interaction during propagation of dengue pseudoinfectious virus in stable CprME-expressing cells.

Dengue virus assembly involves the encapsidation of genomic RNA by the capsid protein (C) and the acquisition of an envelope comprising the premembrane (prM) and envelope (E) glycoproteins. This rapid process, lacking in detectable nucleocapsid intermediates, may impose authentic C-prM-E arrangement as a prerequisite for efficient particle assembly. A mosquito cell-based complementation system was employed in this study to investigate the possibility that expression of the three structural proteins in trans allows the efficient production of a partially C-deleted dengue virus as compared to the presence of C alone. Following the transfection of ΔC56-capped RNA transcripts into C6/36 cells transiently expressing C or CprME, the production of the single-cycle virus was comparable. Subsequent propagation in the stable CprME-expressing clone, however, supported virus adaptation leading to acquisition of the L29P and S101F (PF) dual mutations in the C protein. The triple mutant, ΔC56(PF), exhibited enhanced levels of virus replication, specific infectivity and frequent increases of intracellular C dimer, as compared with ΔC56 in the CprME-clone. The PF mutations were associated with the accumulation of truncated CprM in ΔC56(PF)-infected cells, and uncleaved CprM as well as reduced intracellular C-dimer when the dual mutations were introduced into the wild-type dengue virus genetic background. These results indicate that the PF mutations may exert a replication-enhancing effect for the triple mutant virus by relieving the interference of trans-complementing structural proteins during viral assembly and suggest that the C-prM-E arrangement may be advantageous for pseudoinfectious virus production.