Table 1.
Mutagenic primers.
Table 2.
List of primers used for RT-qPCR.
Table 3.
Primer sequences for detecting HBV DNA and HBV RNA by real-time PCR.
Fig 1.
Detection of N-linked glycosylation in the mutated LHBs.
HEK293T cells were transfected with plasmids expressing either WT LHBs or N-glycan mutants.
Fig 2.
Relative mRNA levels of autophagic markers in HepG2 cells.
The relative mRNA levels of (A) BECN1, (B) ATG9A, (C) ATG12, (D) ATG16L1, (E) AMPK, and (F) mTOR in HepG2 cells were determined 72 hours after transfection with LHBs mutants compared to WT. Nutrient starvation-induced autophagy in HepG2 cells, compared to that in non-starved cells, served as a positive control. β-Actin was used as an internal control. Data are plotted as the mean ± SEM. *p < .05; **p < .01; ***p < .001; #p < .05; ##p < .01; ###p < .001; ns, not significant.
Fig 3.
Relative mRNA levels of autophagic markers in Huh-7 cells.
The relative mRNA levels of (A) BECN1, (B) ATG9A, (C) ATG12, (D) ATG16L1, (E) AMPK, and (F) mTOR in Huh-7 cells were determined 72 hours after transfection with LHBs mutants compared to WT. Nutrient starvation-induced autophagy in HepG2 cells, compared to that in non-starved cells, served as a positive control. β-Actin was used as an internal control. Data are plotted as the mean ± SEM. *p < .05; **p < .01; ***p < .001; #p < .05; ##p < .01; ###p < .001; ns, not significant.
Fig 4.
Effects of wild-type and LHBs mutants on LC3 conversion in transfected cells.
(A) HepG2 cells and (C) Huh-7 cells were either starved or transfected with pcDNA3.1 plasmid (control) or a plasmid expressing the WT or LHBs mutants. Seventy-two hours post-transfection, total cell proteins were collected, and the expression levels of LC3-II were determined by western blotting. GAPDH was used as the protein loading control. The relative intensities of the bands were quantified by normalization to GAPDH using the ImageJ software. Levels of the corresponding mRNAs were determined by RT-qPCR using specific primers (B and D). Data are presented as the mean ± SEM of three independent experiments. *p < .05; **p < .01; ***p < .001; #p < .05; ##p < .01; ###p < .001; ns, not significant.
Fig 5.
Detection and quantification of acidic vesicular organelles.
(A) Immunofluorescence microscopy of acridine orange-stained Huh-7 cells starved for 6 hours with EBSS or transfected with pcDNA3.1 plasmid (control), WT, or LHBs mutants. An increase in the number of cells with AO accumulating acidic vesicular organelles (orange-red fluorescence) was evident. (B) Quantitation of cells with AVO’s from four to six random image fields, totaling approximately 150 cells in each image field. (C) Quantification of the fluorescence intensity in control and LHBs-transfected Huh-7 cells was performed using the ImageJ software. Values represent the mean ± SEM. *p < .05; **p < .01; ***p < .001.
Fig 6.
Impact of wild-type and LHBs mutants on HBV DNA replication, secretion, and gene expression.
(A-B) HepG2.2.15 cells were transfected with plasmids expressing either WT LHBs or N-glycan mutants of LHBs and harvested after 72 hours. (A) Intracellular HBV DNA and (B) HBV progeny DNA from secreted particles were quantified using qPCR. (C-D) HepG2.2.15 cells were harvested 72 hours post-transfection for the detection of the relative expression levels of (C) HBsAg and (D) HBx using RT-qPCR. The data are plotted as mean and SEM. *p < .05; **p < .01; ***p < .001; ns, not significant.
Fig 7.
The effect of autophagy on HBV replication and secretion.
(A-B) HepG2.2.15 cells were transfected with plasmids expressing either WT LHBs or N-glycan mutants of LHBs for 24 hours and further incubated with an autophagy inhibitor 3-MA for 48 hours. (A) These cells were then extracted HBV replicative intermediate from core particles and quantified by qPCR. (B) HBV progeny DNA in the supernatant was extracted and quantified by qPCR. The data are plotted as mean and SEM. *p < .05; **p < .01; ***p < .001.
Fig 8.
Apoptosis markers in HepG2.2.15 cells.
(A) The relative mRNA levels of BAX, BCL2, AIFM1 and Caspase-3 were determined by RT-qPCR and (B) Immunoblot analysis of Caspase-3 in HepG2.2.15 cells were detected following 72 hours transfection. Data are plotted as the mean ± SEM. ns, not significant.