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TGF-β suppression of HBV RNA through AID-dependent recruitment of an RNA exosome complex.

Liang G, Liu G, Kitamura K, Wang Z, Chowdhury S, Monjurul AM, Wakae K, Koura M, Shimadu M, Kinoshita K, Muramatsu M - PLoS Pathog. (2015)

Bottom Line: Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication.Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited.These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; Department of Microbiology and Immunology, Columbia University, New York, New York, United States of America.

ABSTRACT
Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.

No MeSH data available.


Related in: MedlinePlus

Intact P protein is required for AID-mediated downregulation of HBV transcripts and AID associates with HBV P protein.(A) Schematic diagram of wild-type and mutant HBV replicon plasmids. Partially redundant HBV genomic DNA is shown as black boxes and the positions of 5′-ε and 3′-ε are shown. Open reading frames corresponding to C, P, S, and X genes are shown as open boxes. The position of the frame-shift mutation in the mutant replicon plasmid (pPB-ΔP) is indicated as an open triangle. This frame-shift mutation results in loss of the C-terminal portion (polymerase and RNase H domains) from the P protein; pCMV, CMV promoter. (B) Schematic diagram of P protein domain structure; (C) Replicon plasmid (pPB or pPB-ΔP) and GFP fusion expression vectors (mock, AID, and p19-mutant AID) were transfected into Huh7 cells, and after four days, AID-mediated downregulation of HBV transcripts was compared between two replicon plasmids or between wild-type and p19 mutant AID using northern blotting. Expression of HBV core and GFP fusion proteins (mock, AID, and p19-mutant AID) was confirmed using SDS-PAGE and western blotting. (D) Wild-type replicon plasmid (pPB) and indicated protein expression vectors (FLAG-P, FLAG-PΔC, or AID) were transfected into Huh7 cells. Three days later, physical associations between AID and FLAG-P (or FLAG-PΔC) proteins were determined using immunoprecipitation (IP). In lane 9, crude extract was incubated with RNase A before immunoprecipitation. Positions of FLAG-P and FLAG-PΔC proteins are indicated by arrows and diamonds, respectively. The structure of FLAG-PΔC protein is shown in B. Input; crude extract. Data are representative of two to three independent experiments.
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ppat.1004780.g004: Intact P protein is required for AID-mediated downregulation of HBV transcripts and AID associates with HBV P protein.(A) Schematic diagram of wild-type and mutant HBV replicon plasmids. Partially redundant HBV genomic DNA is shown as black boxes and the positions of 5′-ε and 3′-ε are shown. Open reading frames corresponding to C, P, S, and X genes are shown as open boxes. The position of the frame-shift mutation in the mutant replicon plasmid (pPB-ΔP) is indicated as an open triangle. This frame-shift mutation results in loss of the C-terminal portion (polymerase and RNase H domains) from the P protein; pCMV, CMV promoter. (B) Schematic diagram of P protein domain structure; (C) Replicon plasmid (pPB or pPB-ΔP) and GFP fusion expression vectors (mock, AID, and p19-mutant AID) were transfected into Huh7 cells, and after four days, AID-mediated downregulation of HBV transcripts was compared between two replicon plasmids or between wild-type and p19 mutant AID using northern blotting. Expression of HBV core and GFP fusion proteins (mock, AID, and p19-mutant AID) was confirmed using SDS-PAGE and western blotting. (D) Wild-type replicon plasmid (pPB) and indicated protein expression vectors (FLAG-P, FLAG-PΔC, or AID) were transfected into Huh7 cells. Three days later, physical associations between AID and FLAG-P (or FLAG-PΔC) proteins were determined using immunoprecipitation (IP). In lane 9, crude extract was incubated with RNase A before immunoprecipitation. Positions of FLAG-P and FLAG-PΔC proteins are indicated by arrows and diamonds, respectively. The structure of FLAG-PΔC protein is shown in B. Input; crude extract. Data are representative of two to three independent experiments.

Mentions: To investigate the mechanism of AID-mediated downregulation of HBV transcripts, we initially focus on the viral P protein, because AID, P protein and HBV transcripts form RNP complex [26]. In these experiments, we applied a mutant HBV replicon plasmid (pPB-ΔP, Fig 4A) that expresses a mutant P protein lacking the C-terminal half including catalytic DNA polymerase and RNase H domains [26]. Transfection with pPB-ΔP did not support nucleocapsid DNA synthesis due to inhibition of reverse-transcription, although HBV transcription and core protein synthesis remained intact in Huh7 cells (Fig 4C, lanes 1 and 4). AID-mediated downregulation of HBV transcripts was compared between pPB- and pPB-ΔP-transfected Huh7 cells. As shown in Fig 4C, AID-mediated downregulation of HBV transcripts was not observed in pPB-ΔP-transfected Huh7 cells, indicating that AID-mediated downregulation of HBV transcripts requires intact viral P protein.


TGF-β suppression of HBV RNA through AID-dependent recruitment of an RNA exosome complex.

Liang G, Liu G, Kitamura K, Wang Z, Chowdhury S, Monjurul AM, Wakae K, Koura M, Shimadu M, Kinoshita K, Muramatsu M - PLoS Pathog. (2015)

Intact P protein is required for AID-mediated downregulation of HBV transcripts and AID associates with HBV P protein.(A) Schematic diagram of wild-type and mutant HBV replicon plasmids. Partially redundant HBV genomic DNA is shown as black boxes and the positions of 5′-ε and 3′-ε are shown. Open reading frames corresponding to C, P, S, and X genes are shown as open boxes. The position of the frame-shift mutation in the mutant replicon plasmid (pPB-ΔP) is indicated as an open triangle. This frame-shift mutation results in loss of the C-terminal portion (polymerase and RNase H domains) from the P protein; pCMV, CMV promoter. (B) Schematic diagram of P protein domain structure; (C) Replicon plasmid (pPB or pPB-ΔP) and GFP fusion expression vectors (mock, AID, and p19-mutant AID) were transfected into Huh7 cells, and after four days, AID-mediated downregulation of HBV transcripts was compared between two replicon plasmids or between wild-type and p19 mutant AID using northern blotting. Expression of HBV core and GFP fusion proteins (mock, AID, and p19-mutant AID) was confirmed using SDS-PAGE and western blotting. (D) Wild-type replicon plasmid (pPB) and indicated protein expression vectors (FLAG-P, FLAG-PΔC, or AID) were transfected into Huh7 cells. Three days later, physical associations between AID and FLAG-P (or FLAG-PΔC) proteins were determined using immunoprecipitation (IP). In lane 9, crude extract was incubated with RNase A before immunoprecipitation. Positions of FLAG-P and FLAG-PΔC proteins are indicated by arrows and diamonds, respectively. The structure of FLAG-PΔC protein is shown in B. Input; crude extract. Data are representative of two to three independent experiments.
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Related In: Results  -  Collection

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ppat.1004780.g004: Intact P protein is required for AID-mediated downregulation of HBV transcripts and AID associates with HBV P protein.(A) Schematic diagram of wild-type and mutant HBV replicon plasmids. Partially redundant HBV genomic DNA is shown as black boxes and the positions of 5′-ε and 3′-ε are shown. Open reading frames corresponding to C, P, S, and X genes are shown as open boxes. The position of the frame-shift mutation in the mutant replicon plasmid (pPB-ΔP) is indicated as an open triangle. This frame-shift mutation results in loss of the C-terminal portion (polymerase and RNase H domains) from the P protein; pCMV, CMV promoter. (B) Schematic diagram of P protein domain structure; (C) Replicon plasmid (pPB or pPB-ΔP) and GFP fusion expression vectors (mock, AID, and p19-mutant AID) were transfected into Huh7 cells, and after four days, AID-mediated downregulation of HBV transcripts was compared between two replicon plasmids or between wild-type and p19 mutant AID using northern blotting. Expression of HBV core and GFP fusion proteins (mock, AID, and p19-mutant AID) was confirmed using SDS-PAGE and western blotting. (D) Wild-type replicon plasmid (pPB) and indicated protein expression vectors (FLAG-P, FLAG-PΔC, or AID) were transfected into Huh7 cells. Three days later, physical associations between AID and FLAG-P (or FLAG-PΔC) proteins were determined using immunoprecipitation (IP). In lane 9, crude extract was incubated with RNase A before immunoprecipitation. Positions of FLAG-P and FLAG-PΔC proteins are indicated by arrows and diamonds, respectively. The structure of FLAG-PΔC protein is shown in B. Input; crude extract. Data are representative of two to three independent experiments.
Mentions: To investigate the mechanism of AID-mediated downregulation of HBV transcripts, we initially focus on the viral P protein, because AID, P protein and HBV transcripts form RNP complex [26]. In these experiments, we applied a mutant HBV replicon plasmid (pPB-ΔP, Fig 4A) that expresses a mutant P protein lacking the C-terminal half including catalytic DNA polymerase and RNase H domains [26]. Transfection with pPB-ΔP did not support nucleocapsid DNA synthesis due to inhibition of reverse-transcription, although HBV transcription and core protein synthesis remained intact in Huh7 cells (Fig 4C, lanes 1 and 4). AID-mediated downregulation of HBV transcripts was compared between pPB- and pPB-ΔP-transfected Huh7 cells. As shown in Fig 4C, AID-mediated downregulation of HBV transcripts was not observed in pPB-ΔP-transfected Huh7 cells, indicating that AID-mediated downregulation of HBV transcripts requires intact viral P protein.

Bottom Line: Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication.Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited.These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; Department of Microbiology and Immunology, Columbia University, New York, New York, United States of America.

ABSTRACT
Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner.

No MeSH data available.


Related in: MedlinePlus