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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

Transcription dependency for TGF-β1-mediated reduction of HBV transcripts and a proposed model.HBV-expressing 7T7-8 cells were treated with 10 ng/ml TGF-β1 (A) or transfected with AID (or GFP) expression plasmid (B) and cultivated for 3 days. At 18 h before harvest, 100 ng/ml actinomycin D (ActD) was added to block transcription. Total RNA was extracted to measure HBV RNA levels (normalized by HPRT) by qRT-PCR. HBV RNA levels in non-treated (A) and GFP transfected cells (B) were taken as one. **P < 0.01 (t-test); Data are representative of two independent experiments and error bars represent standard errors of the mean. (C) Hypothetical model: Left panel, the canonical HBV life cycle; (a) After the entry of HBV into a hepatocyte, nucleocapsid NC-DNA is converted into cccDNA. (b) Subsequently, cccDNA expresses viral transcripts, including pgRNA, pre-S1, pre-S2/S, X, and pre-C mRNAs. In this study, most viral RNAs were transcribed from the HBV plasmid instead of cccDNA. All transcripts possess the 3′-ε RNA stem-loop structure; only pgRNA is shown. (c) P protein binds to the ε structure and stabilizes it, and the core protein (indicated by hexagons) is then recruited to form the nucleocapsid. (d) Inside the nucleocapsid, P protein reverse transcribes pgRNA to produce NC-DNA. A mature nucleocapsid gains S proteins and is secreted as an infectious virion. The minor fraction of nucleocapsid may enter a second intracellular viral cycle. (e) Right panel, TGF-β1 stimulation of hepatocytes induces AID expression. (f) AID associates with the RNA exosome proteins. The RNA exosome comprises ring-like core and exonuclease catalytic components. AID associates with HBV transcripts and P proteins. (g) Consequently, AID bridges the RNA exosome with the RNP complex of HBV transcripts and P protein, which may trigger the degradation of HBV transcripts.
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ppat.1004780.g008: Transcription dependency for TGF-β1-mediated reduction of HBV transcripts and a proposed model.HBV-expressing 7T7-8 cells were treated with 10 ng/ml TGF-β1 (A) or transfected with AID (or GFP) expression plasmid (B) and cultivated for 3 days. At 18 h before harvest, 100 ng/ml actinomycin D (ActD) was added to block transcription. Total RNA was extracted to measure HBV RNA levels (normalized by HPRT) by qRT-PCR. HBV RNA levels in non-treated (A) and GFP transfected cells (B) were taken as one. **P < 0.01 (t-test); Data are representative of two independent experiments and error bars represent standard errors of the mean. (C) Hypothetical model: Left panel, the canonical HBV life cycle; (a) After the entry of HBV into a hepatocyte, nucleocapsid NC-DNA is converted into cccDNA. (b) Subsequently, cccDNA expresses viral transcripts, including pgRNA, pre-S1, pre-S2/S, X, and pre-C mRNAs. In this study, most viral RNAs were transcribed from the HBV plasmid instead of cccDNA. All transcripts possess the 3′-ε RNA stem-loop structure; only pgRNA is shown. (c) P protein binds to the ε structure and stabilizes it, and the core protein (indicated by hexagons) is then recruited to form the nucleocapsid. (d) Inside the nucleocapsid, P protein reverse transcribes pgRNA to produce NC-DNA. A mature nucleocapsid gains S proteins and is secreted as an infectious virion. The minor fraction of nucleocapsid may enter a second intracellular viral cycle. (e) Right panel, TGF-β1 stimulation of hepatocytes induces AID expression. (f) AID associates with the RNA exosome proteins. The RNA exosome comprises ring-like core and exonuclease catalytic components. AID associates with HBV transcripts and P proteins. (g) Consequently, AID bridges the RNA exosome with the RNP complex of HBV transcripts and P protein, which may trigger the degradation of HBV transcripts.

Mentions: Immunoglobulin gene diversification triggered by AID is coupled with the transcription of immunoglobulin locus [8,9]. Here we examined whether AID-mediated HBV RNA downregulation is also coupled with transcription using a transcription inhibitor actinomycin D (ActD). Using a stable HBV transfectant (7T7-8), we generated experimental conditions in which endogenous or ectopic AID is expressed in HBV-replicating cells. ActD was then added to evaluate whether it could downregulate HBV RNA even in ActD-treated cells. As shown in Fig 8A and 8B, no significant synergistic reduction in HBV RNA levels by ActD and AID was observed in TGF-β1-treated and AID-overexpressing cells, indicating that AID was unable to reduce HBV RNA levels in ActD-treated cells. These results suggest that AID-mediated HBV RNA downregulation depends on transcription, similar to the immunoglobulin gene diversification triggered by AID.


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)

Transcription dependency for TGF-β1-mediated reduction of HBV transcripts and a proposed model.HBV-expressing 7T7-8 cells were treated with 10 ng/ml TGF-β1 (A) or transfected with AID (or GFP) expression plasmid (B) and cultivated for 3 days. At 18 h before harvest, 100 ng/ml actinomycin D (ActD) was added to block transcription. Total RNA was extracted to measure HBV RNA levels (normalized by HPRT) by qRT-PCR. HBV RNA levels in non-treated (A) and GFP transfected cells (B) were taken as one. **P < 0.01 (t-test); Data are representative of two independent experiments and error bars represent standard errors of the mean. (C) Hypothetical model: Left panel, the canonical HBV life cycle; (a) After the entry of HBV into a hepatocyte, nucleocapsid NC-DNA is converted into cccDNA. (b) Subsequently, cccDNA expresses viral transcripts, including pgRNA, pre-S1, pre-S2/S, X, and pre-C mRNAs. In this study, most viral RNAs were transcribed from the HBV plasmid instead of cccDNA. All transcripts possess the 3′-ε RNA stem-loop structure; only pgRNA is shown. (c) P protein binds to the ε structure and stabilizes it, and the core protein (indicated by hexagons) is then recruited to form the nucleocapsid. (d) Inside the nucleocapsid, P protein reverse transcribes pgRNA to produce NC-DNA. A mature nucleocapsid gains S proteins and is secreted as an infectious virion. The minor fraction of nucleocapsid may enter a second intracellular viral cycle. (e) Right panel, TGF-β1 stimulation of hepatocytes induces AID expression. (f) AID associates with the RNA exosome proteins. The RNA exosome comprises ring-like core and exonuclease catalytic components. AID associates with HBV transcripts and P proteins. (g) Consequently, AID bridges the RNA exosome with the RNP complex of HBV transcripts and P protein, which may trigger the degradation of HBV transcripts.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383551&req=5

ppat.1004780.g008: Transcription dependency for TGF-β1-mediated reduction of HBV transcripts and a proposed model.HBV-expressing 7T7-8 cells were treated with 10 ng/ml TGF-β1 (A) or transfected with AID (or GFP) expression plasmid (B) and cultivated for 3 days. At 18 h before harvest, 100 ng/ml actinomycin D (ActD) was added to block transcription. Total RNA was extracted to measure HBV RNA levels (normalized by HPRT) by qRT-PCR. HBV RNA levels in non-treated (A) and GFP transfected cells (B) were taken as one. **P < 0.01 (t-test); Data are representative of two independent experiments and error bars represent standard errors of the mean. (C) Hypothetical model: Left panel, the canonical HBV life cycle; (a) After the entry of HBV into a hepatocyte, nucleocapsid NC-DNA is converted into cccDNA. (b) Subsequently, cccDNA expresses viral transcripts, including pgRNA, pre-S1, pre-S2/S, X, and pre-C mRNAs. In this study, most viral RNAs were transcribed from the HBV plasmid instead of cccDNA. All transcripts possess the 3′-ε RNA stem-loop structure; only pgRNA is shown. (c) P protein binds to the ε structure and stabilizes it, and the core protein (indicated by hexagons) is then recruited to form the nucleocapsid. (d) Inside the nucleocapsid, P protein reverse transcribes pgRNA to produce NC-DNA. A mature nucleocapsid gains S proteins and is secreted as an infectious virion. The minor fraction of nucleocapsid may enter a second intracellular viral cycle. (e) Right panel, TGF-β1 stimulation of hepatocytes induces AID expression. (f) AID associates with the RNA exosome proteins. The RNA exosome comprises ring-like core and exonuclease catalytic components. AID associates with HBV transcripts and P proteins. (g) Consequently, AID bridges the RNA exosome with the RNP complex of HBV transcripts and P protein, which may trigger the degradation of HBV transcripts.
Mentions: Immunoglobulin gene diversification triggered by AID is coupled with the transcription of immunoglobulin locus [8,9]. Here we examined whether AID-mediated HBV RNA downregulation is also coupled with transcription using a transcription inhibitor actinomycin D (ActD). Using a stable HBV transfectant (7T7-8), we generated experimental conditions in which endogenous or ectopic AID is expressed in HBV-replicating cells. ActD was then added to evaluate whether it could downregulate HBV RNA even in ActD-treated cells. As shown in Fig 8A and 8B, no significant synergistic reduction in HBV RNA levels by ActD and AID was observed in TGF-β1-treated and AID-overexpressing cells, indicating that AID was unable to reduce HBV RNA levels in ActD-treated cells. These results suggest that AID-mediated HBV RNA downregulation depends on transcription, similar to the immunoglobulin gene diversification triggered by AID.

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