BackgroundDengue 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.FindingsUsing 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.ConclusionThe 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.

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Research over the past few years showed that mitochondrial morphology is a key player in regulation of vital cell physiological processes right from respiration to regulation of apoptosis. Recently the role of mitochondria in virus morphogenesis is gaining importance. In this study, the interaction of dengue virus (DENV) with mitochondria during its replication was investigated, with special emphasis on the association of specific viral proteins with mitochondria. By live cell imaging, it was seen that within an hour of infection, mitochondria showed rapid fission and fusion, indicative of high energy state in the cells. This was followed by recruitment of mitochondria on microtubules to perinuclear region and colocalization with viral factories. As infection progressed, there was circularization and disintegration of mitochondria. In conclusion, our observations suggest a basic dichotomy in the functioning of mitochondria in replication of DENV. Initially, during replication, mitochondria provide energy to ongoing virus replication; while at later stages cells are characterized by mitochondrial dysfunction leading to apoptosis. Of special interest is the implication of viral core protein in induction of apoptosis.The present study is supported by CSIR (F.NO. 9/698(9)/2004‐EMR‐I) and ICMR, India.