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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

TGF-β1-mediated downregulation of HBV transcripts requires RNA exosome proteins.Huh7 cells were transfected with pPB and indicated siRNAs. Six hours after transfection, the cells were incubated in the presence or absence of 10-ng/mL TGF-β1 for 3 days. Total RNA was analyzed using northern blotting (A) and qRT-PCR to determine HBV transcription of AID, Exosc3, and Exosc6 (B, D, E, F). In C, NC-DNA from secreted virions was also measured by qPCR. Transfection of siAID and siExosc3 partially restored TGF-β1-mediated downregulation of HBV transcripts and viral production; *P < 0.05, **P < 0.01 (t-test); error bars represent standard errors of the mean. Data are representative of two to three independent experiments.
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ppat.1004780.g006: TGF-β1-mediated downregulation of HBV transcripts requires RNA exosome proteins.Huh7 cells were transfected with pPB and indicated siRNAs. Six hours after transfection, the cells were incubated in the presence or absence of 10-ng/mL TGF-β1 for 3 days. Total RNA was analyzed using northern blotting (A) and qRT-PCR to determine HBV transcription of AID, Exosc3, and Exosc6 (B, D, E, F). In C, NC-DNA from secreted virions was also measured by qPCR. Transfection of siAID and siExosc3 partially restored TGF-β1-mediated downregulation of HBV transcripts and viral production; *P < 0.05, **P < 0.01 (t-test); error bars represent standard errors of the mean. Data are representative of two to three independent experiments.

Mentions: To further confirm that the RNA exosome is involved in AID-mediated downregulation of HBV transcripts, we used the siRNA knockdown of Exosc3, which is essential for the RNA exosome function [32]. In these experiments, siRNAs against Exosc3 were co-transfected with the HBV plasmid and AID (or GFP) expression vectors, and HBV replication was determined. Northern blotting, NAGE assays, and qRT-PCR analyses showed the attenuation of AID-mediated downregulation of HBV transcripts and nucleocapsid formation in siExosc3 transfectants (Fig 5E–5G). In contrast, AID, GFP, and GAPDH expression were not affected by Exosc3 depletion (Fig 5E, bottom). Importantly, knock down of Exosc3 did not increase HBV RNA levels in GFP transfected samples. Moreover, siExosc3 transfection attenuated TGF-β1-mediated downregulation of HBV transcripts and nucleocapsid formation in a similar manner to that observed after transfection with siAID (Fig 6A–6F). In further experiments, knockdown of another RNA exosome component Exosc6 also attenuated TGF-β1-mediated downregulation of HBV transcripts and nucleocapsid formation, albeit less effectively than the knockdown of siExosc3 and AID (Fig 6A–6F). Similarly, the contributions of AID and Exosc3 to TGF-β1-mediated downregulation of HBV transcripts were examined in stably HBV-transfected Huh7 cells (7T7-8) [26]. The short hairpin (sh) RNA expressing lentivirus was transduced into 7T7-8 cells, and two stable transfectants (shAID and shExosc3) and a control transfectant (shLuc) were established after puromycin selection. These cells were then cultured in the presence or absence of TGF-β1 (Fig 7A). Subsequent qRT-PCR and western blotting showed reduced endogenous AID and Exosc3 expression (Fig 7B–7E). Comparison of HBV transcript levels between TGF-β1-treated and non-treated 7T7-8 cells revealed that TGF-β1-mediated reduction of HBV transcripts is restored by the knockdown of AID and Exosc3 (Fig 7F). Taken together, these data indicate that RNA exosome proteins (Exosc3 and Exosc6) and AID are required for TGF-β1-mediated downregulutation of HBV transcripts.


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)

TGF-β1-mediated downregulation of HBV transcripts requires RNA exosome proteins.Huh7 cells were transfected with pPB and indicated siRNAs. Six hours after transfection, the cells were incubated in the presence or absence of 10-ng/mL TGF-β1 for 3 days. Total RNA was analyzed using northern blotting (A) and qRT-PCR to determine HBV transcription of AID, Exosc3, and Exosc6 (B, D, E, F). In C, NC-DNA from secreted virions was also measured by qPCR. Transfection of siAID and siExosc3 partially restored TGF-β1-mediated downregulation of HBV transcripts and viral production; *P < 0.05, **P < 0.01 (t-test); error bars represent standard errors of the mean. Data are representative of two to three independent experiments.
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ppat.1004780.g006: TGF-β1-mediated downregulation of HBV transcripts requires RNA exosome proteins.Huh7 cells were transfected with pPB and indicated siRNAs. Six hours after transfection, the cells were incubated in the presence or absence of 10-ng/mL TGF-β1 for 3 days. Total RNA was analyzed using northern blotting (A) and qRT-PCR to determine HBV transcription of AID, Exosc3, and Exosc6 (B, D, E, F). In C, NC-DNA from secreted virions was also measured by qPCR. Transfection of siAID and siExosc3 partially restored TGF-β1-mediated downregulation of HBV transcripts and viral production; *P < 0.05, **P < 0.01 (t-test); error bars represent standard errors of the mean. Data are representative of two to three independent experiments.
Mentions: To further confirm that the RNA exosome is involved in AID-mediated downregulation of HBV transcripts, we used the siRNA knockdown of Exosc3, which is essential for the RNA exosome function [32]. In these experiments, siRNAs against Exosc3 were co-transfected with the HBV plasmid and AID (or GFP) expression vectors, and HBV replication was determined. Northern blotting, NAGE assays, and qRT-PCR analyses showed the attenuation of AID-mediated downregulation of HBV transcripts and nucleocapsid formation in siExosc3 transfectants (Fig 5E–5G). In contrast, AID, GFP, and GAPDH expression were not affected by Exosc3 depletion (Fig 5E, bottom). Importantly, knock down of Exosc3 did not increase HBV RNA levels in GFP transfected samples. Moreover, siExosc3 transfection attenuated TGF-β1-mediated downregulation of HBV transcripts and nucleocapsid formation in a similar manner to that observed after transfection with siAID (Fig 6A–6F). In further experiments, knockdown of another RNA exosome component Exosc6 also attenuated TGF-β1-mediated downregulation of HBV transcripts and nucleocapsid formation, albeit less effectively than the knockdown of siExosc3 and AID (Fig 6A–6F). Similarly, the contributions of AID and Exosc3 to TGF-β1-mediated downregulation of HBV transcripts were examined in stably HBV-transfected Huh7 cells (7T7-8) [26]. The short hairpin (sh) RNA expressing lentivirus was transduced into 7T7-8 cells, and two stable transfectants (shAID and shExosc3) and a control transfectant (shLuc) were established after puromycin selection. These cells were then cultured in the presence or absence of TGF-β1 (Fig 7A). Subsequent qRT-PCR and western blotting showed reduced endogenous AID and Exosc3 expression (Fig 7B–7E). Comparison of HBV transcript levels between TGF-β1-treated and non-treated 7T7-8 cells revealed that TGF-β1-mediated reduction of HBV transcripts is restored by the knockdown of AID and Exosc3 (Fig 7F). Taken together, these data indicate that RNA exosome proteins (Exosc3 and Exosc6) and AID are required for TGF-β1-mediated downregulutation of HBV transcripts.

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