Insights into the Internalization and Retrograde Trafficking of Dengue 2 Virus in BHK-21 Cells

Abstract

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.

Inhibition of N‐myristoyltransferase1 affects dengue virus replication

“…; Thepparit et al., ) to examine NMT1 gene expression in human dendritic cells at different time‐points postinfection. The results revealed the significant upregulation of NMT1 at 1, 12, and 36 hr, which might represent the time period of DENV infection, and which suggests NMT‐related membrane‐associated viral entry (Abou‐Jaoude, Molina, Maurel, & Sureau, ; Maurer‐Stroh & Eisenhaber, ; Thaa et al., ), transcription and endoplasmic reticulum involvement (Moriya et al., ), and viral exocytosis (Figure ).…”

“…The cells were harvested at specific experiment time-points according to the viral life cycle processes, consisting of adsorption, viral fusion, protein translation/ genome replication, viral assembly, viral maturation, and viral release (Mukhopadhyay, Kuhn, & Rossmann, 2005). We harvested the cells at 1, 12, and 36 hr postinfection, as adjusted from previously described studies (Barth, 1992;Mosso, Galvan-Mendoza, Ludert, & del Angel, 2008;Shrivastava, Sripada, Kaur, Shah, & Cecilia, 2011;Thepparit, Phoolcharoen, Suksanpaisan, & Smith, 2004). (Grand, 1989;Hulo et al, 2006;Towler, Gordon, Adams, & Glaser, 1988).…”

A plasmid-based reporter system for live cell imaging of dengue virus infected cells

“…Additionally, the ability to monitor infection by fluorescence relocalization in living cells also suggests the possibility of continuous visualization of DENV infection. The 4B5-EGFP reporter was proteolytically processed as early as 8 hrs post-infection, which correlates with synthesis of new viral protein (Shrivastava et al, 2011). The ability to detect viral infection during initial translation of the viral genome will extend analysis of cellular events to early time points.…”

Molecular Mechanisms Involved in Antibody‐Dependent Enhancement of Dengue Virus Infection in Humans

“…In addition to acidification, microtubule integrity and the transport proteins kinesin and dynein are important factors in endosome maturation. Disruption of microtubules in ECV306 cells did not appear to inhibit infection , whereas in BHK‐21 cells DENV2 was found to associate with dynein . Using single‐particle tracking, both microtubule‐dependent and ‐independent intracellular transport behaviour of DENV was seen in BS‐C‐1 cells prior to membrane fusion.…”