Insights into the internalization and retrograde trafficking of Dengue 2 virus in BHK-21 cells.

BACKGROUND: Dengue virus (DENV) enters cells via endocytosis, traffics to perinuclear (PN) region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT) and dynein in the early events of DENV replication. FINDINGS: Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E) protein of the virion associated with dynamin II from 0-30 min post infection (p.i.). The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN) region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h-8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24-48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation. CONCLUSION: The study shows for the first time the association of dynamin II with DENV-2 during entry and dynein dependent retrograde trafficking of DENV proteins on microtubules