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Hepatitis B virus-specific miRNAs and Argonaute2 play a role in the viral life cycle.

Hayes CN, Akamatsu S, Tsuge M, Miki D, Akiyama R, Abe H, Ochi H, Hiraga N, Imamura M, Takahashi S, Aikata H, Kawaoka T, Kawakami Y, Ohishi W, Chayama K - PLoS ONE (2012)

Bottom Line: AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments.HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments.These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT

Unlabelled: Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion: These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.

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Related in: MedlinePlus

Co-localization of HBcAg and HBsAg with AGO2 in stably transfected T23 cells.A) Anti-AGO2 and anti-HBc staining overlapped in stably transfected T23 cells, but not in HepG2 control cells, suggesting an interaction between HBc and AGO2. B) HBc-AGO2 was detected in T23 but not HepG2 cells using proximity ligation assays (PLA), suggesting a protein-protein interaction between HBcAg and AGO2. C) Overlap of anti-AGO2 and anti-HBs staining suggests co-localization of HBs and AGO2. D) Anti- HBc, and anti-HBs staining overlapped in T23 cells, which may indicate that HBc and HBs co-localize. E) Overlap of anti-AGO2, anti-HBc, and anti-HBs staining in T23 cells suggests that all three proteins may co-localize.
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pone-0047490-g001: Co-localization of HBcAg and HBsAg with AGO2 in stably transfected T23 cells.A) Anti-AGO2 and anti-HBc staining overlapped in stably transfected T23 cells, but not in HepG2 control cells, suggesting an interaction between HBc and AGO2. B) HBc-AGO2 was detected in T23 but not HepG2 cells using proximity ligation assays (PLA), suggesting a protein-protein interaction between HBcAg and AGO2. C) Overlap of anti-AGO2 and anti-HBs staining suggests co-localization of HBs and AGO2. D) Anti- HBc, and anti-HBs staining overlapped in T23 cells, which may indicate that HBc and HBs co-localize. E) Overlap of anti-AGO2, anti-HBc, and anti-HBs staining in T23 cells suggests that all three proteins may co-localize.

Mentions: Using immunocytochemistry and PLA analysis, we found that HBV core protein and AGO2 co-localized within T23 cells (Fig. 1A–B), suggesting a potential protein-protein interaction between HBcAg and AGO2. AGO2 also co-localized with HBs in T23 cells (Fig. 1C), indicating a potential interaction between HBs and AGO2. Overlap between anti-HBc and anti-HBs staining (Fig. 1D) and between anti-AGO2, anti-HBc, and anti-HBs (Fig. 1E) suggests that these three proteins may co-localize. No overlap was observed between anti-AGO2 and anti-HBx staining in HepG2 cells transfected with HBx expression plasmid (p3FLAG-HBx) nor in control cells, suggesting that HBx does not interact with AGO2 (data not shown).


Hepatitis B virus-specific miRNAs and Argonaute2 play a role in the viral life cycle.

Hayes CN, Akamatsu S, Tsuge M, Miki D, Akiyama R, Abe H, Ochi H, Hiraga N, Imamura M, Takahashi S, Aikata H, Kawaoka T, Kawakami Y, Ohishi W, Chayama K - PLoS ONE (2012)

Co-localization of HBcAg and HBsAg with AGO2 in stably transfected T23 cells.A) Anti-AGO2 and anti-HBc staining overlapped in stably transfected T23 cells, but not in HepG2 control cells, suggesting an interaction between HBc and AGO2. B) HBc-AGO2 was detected in T23 but not HepG2 cells using proximity ligation assays (PLA), suggesting a protein-protein interaction between HBcAg and AGO2. C) Overlap of anti-AGO2 and anti-HBs staining suggests co-localization of HBs and AGO2. D) Anti- HBc, and anti-HBs staining overlapped in T23 cells, which may indicate that HBc and HBs co-localize. E) Overlap of anti-AGO2, anti-HBc, and anti-HBs staining in T23 cells suggests that all three proteins may co-localize.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472984&req=5

pone-0047490-g001: Co-localization of HBcAg and HBsAg with AGO2 in stably transfected T23 cells.A) Anti-AGO2 and anti-HBc staining overlapped in stably transfected T23 cells, but not in HepG2 control cells, suggesting an interaction between HBc and AGO2. B) HBc-AGO2 was detected in T23 but not HepG2 cells using proximity ligation assays (PLA), suggesting a protein-protein interaction between HBcAg and AGO2. C) Overlap of anti-AGO2 and anti-HBs staining suggests co-localization of HBs and AGO2. D) Anti- HBc, and anti-HBs staining overlapped in T23 cells, which may indicate that HBc and HBs co-localize. E) Overlap of anti-AGO2, anti-HBc, and anti-HBs staining in T23 cells suggests that all three proteins may co-localize.
Mentions: Using immunocytochemistry and PLA analysis, we found that HBV core protein and AGO2 co-localized within T23 cells (Fig. 1A–B), suggesting a potential protein-protein interaction between HBcAg and AGO2. AGO2 also co-localized with HBs in T23 cells (Fig. 1C), indicating a potential interaction between HBs and AGO2. Overlap between anti-HBc and anti-HBs staining (Fig. 1D) and between anti-AGO2, anti-HBc, and anti-HBs (Fig. 1E) suggests that these three proteins may co-localize. No overlap was observed between anti-AGO2 and anti-HBx staining in HepG2 cells transfected with HBx expression plasmid (p3FLAG-HBx) nor in control cells, suggesting that HBx does not interact with AGO2 (data not shown).

Bottom Line: AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments.HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments.These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT

Unlabelled: Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion: These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.

Show MeSH
Related in: MedlinePlus