Fig 2: Depletion of Rab33B does not affect hepatitis B virus (HBV) subviral empty envelope particles (SVP) release or cell morphology. (A) HuH-7 cells were treated with control small interfering RNA (siCon) or a pool of four siRNA duplexes directed against Rab33B. After 48 h, cells were retransfected with an hemagglutinin (HA)-tagged version of the HBV S envelope gene (S.HA), and lysates and supernatants were harvested 72 h later. To probe for the knockdown efficiency, lysates were immunoblotted with anti-Rab33B antibodies (#6F4). An unspecific band stained by the antibody served as a loading control (Load). Synthesis (Cell) and secretion (Medium) of S.HA were assayed by HA-specific Western blot (WB). The non-glycosylated (p24) and glycosylated forms (gp27, gp28) of S.HA are depicted. Numbers to the right refer to molecular weight standards in kDa. The degree of S.HA release was quantified by densitometric analysis of Western blots and was demonstrated in percent amount relative to the control cells (n = 3, mean ± standard deviation [SD]). (B) HuH-7 cells were transfected with siCon- or siRab33B-specific duplexes for four days and cell lysates were immunoblotted with anti-Rab33B (#sc-271199) and anti-ß-actin antibodies. (C) For immunofluorescence (IF) analyses, cells were fixed and permeabilized using methanol. This fixation method was chosen because the ß-actin antibody is not reactive upon paraformaldehyde (PFA) fixation, as instructed by the supplier. Cells were stained with antibodies against calnexin (CNX), protein disulfide isomerase (PDI), golgin-97, and ß-actin followed by staining with fluorophor-labeled antibodies. DNA staining is shown in blue. Scale bar (C): 10 µm.

Fig 3: Depletion of Rab33B blocks HBV formation and release. (A) HuH-7 cells were treated with control small interfering RNA (siCon) or the Rab33B-specific small interfering RNA (siRab33B) pool for 48 h and were retransfected with with an HBV replicon construct (HBV). After 72 h, lysates and supernatants were harvested. To probe for Rab33B depletion, lysates were immunoblotted with an anti-Rab33B antibody (#6F4). Uniformity of sample loading is shown by an unspecific band stained by the antibody (Load). HBV release was detected by envelope-specific immunoprecipitation of supernatants and real-time PCR of the viral genomes (Virion). Nonenveloped cytoplasmic nucleocapsids (NC) were immunoprecipitated with anti-capsid antibodies and analyzed by PCR. PCR results were demonstrated in percent amount relative to the control-transfected cells. Error bars indicate the standard deviations from the mean of four experiments measured in duplicates. RNA interference (RNAi) effects on the expression of L and core were analyzed by specific WB of lysates prepared with 0.2% Triton X-100. The non-glycosylated (p39) and glycosylated forms (gp42) of L are depicted. Numbers to the right refer to molecular weight standards in kDa. * P < 0.05, ** P < 0.01 compared to control. (B) Total protein concentrations of lysates of siCon- or siRab33B-treated cells were determined using a Bradford assay and demonstrated in percent amount relative to the control cells (n = 2, mean ± SD). (C) To probe for cell lysis, supernatants of transfected cells were assayed for lactate dehydrogenase (LDH) activity (n = 2, mean ± SD). (D) To quantitate the levels of the HBV envelope proteins, lysates and supernatants of transfected cells were analyzed with a HBsAg-specific ELISA (n = 2, mean ± SD).

Fig 4: Effects of Rab33B depletion and reconstitution. HuH-7 cells were treated with control small interfering RNA (siCon) or the Rab33B-specific small interfering RNA (siRab33B) pool for 48 h and were retransfected for 72 h with HBV in the absence or presence of a mouse Myc-tagged Rab33B rescue construct (Rescue) at a 2:0.8 DNA weight ratio, respectively. Cell lysates were assayed by Rab33B-, L envelope protein-, Core-, ß-actin-, Atg16L1-, and Atg5-specific immunoblotting. Experiments were done in duplicate and representative images are shown. Numbers to the right refer to molecular weight standards in kDa.

Fig 5: Effects of Rab33B inactivation on intracellular HBV protein distribution. For RNAi, HuH-7 cells were treated with control or Rab33B-specific siRNAs for two days and were retransfected for two days with pHBV or pHBV?Env, as indicated to the left of the images. (A) To probe for depletion, cells were lysed and assayed by Rab33B- (#sc-271199) and ß-actin-specific WB. (B,C) For IF, cells were fixed and permeablized with PFA and Triton X-100. Cells were immunostained with anti-core (Dako), anti-L (MA18/7), or anti-calnexin (CNX) antibodies followed by staining with AlexaFluor 546-conjugated anti-rabbit and AlexaFluor 488-conjugated anti-mouse antibodies. Core and CNX staining is in red, L staining in green, and DNA staining of the nuclei is in blue. Scale bar: 10 µm. Merge images are shown in the right panels. Outlined areas are shown at larger magnifications.