Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Mar 2;107(9):4383-8.
doi: 10.1073/pnas.0911373107. Epub 2010 Feb 8.

The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication

Donna Sir  1 , Yongjun Tian, Wen-ling Chen, David K Ann, Tien-Sze Benedict Yen, Jing-Hsiung James Ou
Affiliations

The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication

Donna Sir et al. Proc Natl Acad Sci U S A. .

Abstract

Autophagy is a catabolic process by which cells remove long-lived proteins and damaged organelles for recycling. Viral infections may also induce autophagic response. Here we show that hepatitis B virus (HBV), a pathogen that chronically infects approximately 350 million people globally, can enhance autophagic response in cell cultures, mouse liver, and during natural infection. This enhancement of the autophagic response is not coupled by an increase of autophagic protein degradation and is dependent on the viral X protein, which binds to and enhances the enzymatic activity of phosphatidylinositol 3-kinase class III, an enzyme critical for the initiation of autophagy. Further analysis indicates that autophagy enhances HBV DNA replication, with minimal involvement of late autophagic vacuoles in this process. Our studies thus demonstrate that a DNA virus can use autophagy to enhance its own replication and indicate the possibility of targeting the autophagic pathway for the treatment of HBV patients.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Induction of autophagic vacuoles by HBV. (A) Confocal microscopy of Huh7.5–GFP–LC3 cells transfected by the 1.3mer HBV genomic DNA. Cells were stained with the antibody that recognized both HBV core protein and e antigen (HBc/eAg; red) 48 h after DNA transfection. Note that the GFP–LC3 puncta (green) are apparent only in the HBV-positive cell, and a significant overlap of the signals of HBV c/e antigens and GFP puncta is also visible when the images are merged. DAPI (blue) stains for the nuclei. (B) Percentage of cells with GFP–LC3 puncta in nontransfected cells (NC), pUC19-transfected cells, 1.3mer HBV DNA-transfected cells, and nutrient-starved cells. GFP-positive cells were defined as cells that display bright punctate staining. Approximately 50 cells were counted and the experiment was repeated at least three times. Asterisks indicate statistical significance (P < 0.01). (C) Western blot analysis of LC3. Lanes 1 and 2 were Huh7.5 cells transfected with pUC19 and the 1.3mer HBV DNA, respectively. Lane 3 was the naive HepG2 cells and lane 4 was a stable HepG2 cell line that contained replicating HBV DNA (24). Tg08 (lane 6) and Tg05 (lane 8) were two independent transgenic mouse lines that carried the 1.3mer HBV genome (8). Their HBV-negative control littermates were shown in lanes 5 and 7, respectively. Lanes 9 and 10 were liver tissues from HBV noninfected and infected patients, respectively. (Top) LC3 proteins. (Middle) HBV core protein. (Bottom) Actin control.
Fig. 2.
Fig. 2.
Enhancement of autophagic flux by HBV without increasing autophagic protein degradation. (A) Confocal microscopy of HBV surface antigens (HBsAg) (blue), LAMP1 (red), and GFP–LC3 puncta (green) in Huh7.5 cells. (Top) Nutrient-starved cells. (Middle) pUC19-transfected cells. (Bottom) 1.3mer HBV DNA-transfected cells. (B) Long-lived protein degradation assay. Gray + black bar, overall protein degradation rate in the absence of BAF (−BAF); black bar, protein degradation rate in the presence of BAF (+BAF). The gray area of the bars represents the BAF-sensitive protein degradation rate. The results represent the average of three independent experiments. The asterisk indicates statistical significance (P < 0.05).
Fig. 3.
Fig. 3.
Enhancement of HBV DNA replication by autophagy. Huh7.5 cells transfected with pUC19 (lane 1) or the 1.3mer HBV DNA (lanes 2 and 3) were used for the analysis. (A) Western blot analysis of the HBV core protein (Top), LC3 (Middle), and actin (Bottom). Cells transfected with the DNA for 24 h were further treated without (lanes 1 and 2) or with (lane 3) 10 mM 3-MA for 16 h. (B) Southern blot analysis of HBV DNA. Cells without or with 3-MA treatment as in A were lysed for the purification of replicated HBV DNA for the Southern blot analysis. RC, HBV relaxed circular DNA; RI, HBV replicative intermediates. (C) Western blot analysis of Vps34. DNA-transfected cells were further transfected with the negative control (NC), siRNA (lanes 1 and 2), or Vps34 siRNA (lane 3) for 48 h. (D) Southern blot analysis of replicated HBV DNA in cells treated with control or Vps34 siRNA. (E) Western blot analysis of Atg7 expressed in cells transfected with control or Atg7 siRNA. (F) Southern blot analysis of replicated HBV DNA in cells treated with control or Atg7 siRNA.
Fig. 4.
Fig. 4.
Analysis of the autophagic effect on HBV RNAs. Huh7.5 cells were transfected with pUC19 or the 1.3mer HBV genome and treated with 3-MA or siRNA as described in the legend to Fig. 3. (A and B) Northern blot analysis of HBV RNAs. (C and D) Primer extension analysis of total HBV precore and core RNAs in cells (lanes 1–3) or the core RNA packaged in core particles (lanes 4–6). S, S gene transcripts; C, C gene transcripts.
Fig. 5.
Fig. 5.
Effects of HBx on autophagy and HBV DNA replication. (A) Huh7.5 cells were transfected with pUC19 (lane 1), 1.3mer HBV DNA (lane 2), or 1.3mer HBVX− DNA (lane 3) followed by Western blot analysis using anti-LC3, anti-HBV core, or anti-actin antibodies. (B) Western blot analysis of mouse liver lysates. Lane 1, control nontransgenic mouse; lane 2, Tg05 mouse that carried the wild-type 1.3mer HBV genome; and lane 3, Tg38 mouse that carried the X-negative 1.3mer HBVX− genome. (C) Huh7.5 cells were transfected with the control pECE1 vector (lane 1) or pECE1-HAX (lane 2) followed by Western blot analysis using anti-LC3 (Top), anti-HA (Middle), or anti-actin (Bottom) antibodies. (D) Huh7 cells were transfected with pUC19 (lane 1), 1.3mer HBV DNA (lanes 2 and 3), or p1.3mer HBVX− (lanes 4 and 5) and then treated without or with 3-MA for 16 h. Cells were then lysed for Southern blot analysis for HBV DNA. (E) The same experiment as shown in D was repeated using HepG2 cells.
Fig. 6.
Fig. 6.
Physical and functional interactions between HBx and PI3KC3. (A) Coimmunoprecipitation experiments. Huh7.5 cells were transfected with pECE1 (−) or pECE1-HAX (+). After 48 h, cells were lysed and immunoprecipitated with rabbit anti-Vps34 (lanes 1 and 2), control rabbit IgG (lanes 3 and 4), control mouse IgG (lanes 5 and 6), or the mouse anti-HA antibody (lanes 7 and 8). The immunoprecipitated samples were then analyzed by Western blot for Vps34 (Top) and HAX (Middle). Input, total cell lysates. (B) Colocalization of HAX and Flag-tagged PI3KC3 (Vps34). Huh7.5 cells were cotransfected with pFlag–PI3KC3 and pECE1–HAX (Upper) or pECE1 (Lower). Cells were then stained for Flag-tagged PI3KC3 (green) or HAX (red) and analyzed by confocal microscopy. (C) Induction of PI3P by HBx. Huh7.5 cells were cotransfected with p40PX–EGFP and pECE1–HAX (Upper) or its control vector pECE1 (Lower). After 48 h, cells were fixed, stained for HAX (red), and analyzed for the intensity of EGFP (green) by fluorescence microscopy. (D) Induction of PI3P by wild-type HBV but not the X-negative HBV. Huh7.5 cells were cotransfected with p40PX–EGFP and 1.3mer HBV DNA (Top), 1.3mer HBVX− mutant DNA (Middle), or pUC19 (Bottom). Cells were then fixed, stained for HBV core/e antigen, and analyzed by fluorescence microscopy. (E) Histogram of the relative intensities of EGFP shown in Fig. 6 C and D. The EGFP pixel densities of more than 50 cells were measured and that of pUC19-transfected cells was arbitrarily defined as one. The results represent the average of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schmid D, Dengjel J, Schoor O, Stevanovic S, Münz C. Autophagy in innate and adaptive immunity against intracellular pathogens. J Mol Med. 2006;84:194–202. - PubMed
    1. Deretic V, Levine B. Autophagy, immunity, and microbial adaptations. Cell Host Microbe. 2009;5:527–549. - PMC - PubMed
    1. Jackson WT, et al. Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol. 2005;3:e156. - PMC - PubMed
    1. Prentice E, Jerome WG, Yoshimori T, Mizushima N, Denison MR. Coronavirus replication complex formation utilizes components of cellular autophagy. J Biol Chem. 2004;279:10136–10141. - PMC - PubMed
    1. Sir D, et al. Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response. Hepatology. 2008;48:1054–1061. - PMC - PubMed

Publication types