The large GTPase dynamin is required for hepatitis B virus protein secretion from hepatocytes  Ahmad S. Abdulkarim, Hong Cao, Bing Huang, Mark A. McNiven  Journal of Hepatology  Volume 38, Issue 1, Pages 76-83 (January 2003) DOI: 10.1016/S0168-8278(02)00326-4

Fig. 1 Dyn2 function is necessary for the transport of HBV proteins. HepG2.2.15 cells transfected with either wild-type Dyn2 or Dyn2K44A were costained for dynamin and the HBsAg or HBeAg. Wild-type Dyn2 (a,c) had no effect on HBsAg (a′) or HBeAg (c′) distribution. However, expression of Dyn2K44A (b,d) induced an accumulation of the HBsAg (b′) and HBeAg (d′) in a perinuclear region (arrows). Nuclei are circled. Bar: 10 μm. Journal of Hepatology 2003 38, 76-83DOI: (10.1016/S0168-8278(02)00326-4)

Fig. 2 HBsAg accumulates in a perinuclear region in Dyn2K44A-expressing cells. The fluorescence intensity of the HBsAg in HepG2.2.15 cells transfected with wild-type Dyn2 or Dyn2K44A was measured in a perinuclear region using a box of defined size (a). The box was placed on a cell area containing the perinuclear region where viral protein had accumulated. A pixel intensity value for the area within the box was calculated. The fluorescence intensity of HBsAg staining was measured in 100 cells for each construct transfected, Dyn2 and Dyn2K44A. As shown by graphing the averaged fluorescence intensity values (b), there was an increase in the amount of HBsAg in the perinuclear region in Dyn2K44A-expressing cells in comparison to wild-type Dyn2-expressing cells. Similar results were obtained for HBeAg (data not shown). Journal of Hepatology 2003 38, 76-83DOI: (10.1016/S0168-8278(02)00326-4)

Fig. 3 Secretion of the HBsAg and HBeAg is dependent on functional Dyn2. An equal number of HepG2.2.15 cells were transfected with either wild-type Dyn2 or Dyn2K44A constructs. The cell culture supernatant was collected 48 h post-transfection and the HBsAg (a) and HbeAg (b) levels were measured. There was a reduction in the amount of viral protein secreted in Dyn2K44A-transfected cells compared to wild-type Dyn2-transfected cells. Results are based on the average of at least three separate experiments. Journal of Hepatology 2003 38, 76-83DOI: (10.1016/S0168-8278(02)00326-4)

Fig. 4 EM analysis of viral particles trapped in an undefined compartment by Dyn2K44A. HepG2.2.15 cells were transfected with Dyn2K44A-GFP. The localization of GFP-expressing cells was determined using fluorescence microscopy before processing cells for EM. Positive cells were then sectioned and imaged at 5000× (a), 7000× (b), or 20 000× (c,d) magnification using EM. Distinct cytoplasmic vacuoles containing numerous viral particles were detected in Dyn2K44A-expressing cells (b,d). Note the large amount of viral particles contained in some vacuoles (b,d,arrows). Bars: 1.0 μm (a), 0.2 μm (b–d). Journal of Hepatology 2003 38, 76-83DOI: (10.1016/S0168-8278(02)00326-4)

Fig. 5 Mutant dynamin increases the percentage of cytoplasmic vacuoles containing viral particles and the number of viral particles per vacuole. The total number of vacuoles in HepG2.2.15 cells transfected with either GFP-vector, Dyn2-GFP, or Dyn2K44A-GFP and imaged by EM was counted in seven separate cells for each construct. Also, the number of vacuoles containing viral particles was calculated as a percentage of the total vacuoles present as shown in (a). In Dyn2K44A-GFP-transfected cells, a greater percentage of these vacuoles contained viral particles, in comparison to GFP-vector- and Dyn2-GFP-transfected cells. In (b) the average number of viral particles per vacuole of the three different groups was counted (100 vacuoles per group). The number of viral particles per vacuole in Dyn2K44A-GFP-transfected cells was significantly higher than GFP-vector- and Dyn2-GFP-transfected cells. Journal of Hepatology 2003 38, 76-83DOI: (10.1016/S0168-8278(02)00326-4)