TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

<div><p>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.</p></div

IgA switching activity correlates with reduction of HBV transcripts in B cells.

<p>(A, B, C, D) pPB and GFP expression vectors were transiently co-transfected into a mouse B cell line (CH12F3-2). Six hours after transfection, cells were divided into two groups and stimulated with (or without) CD40 ligand, IL-4, and TGF-β1 (CIT) for 3 days to induce IgA switching; (A) Schematic diagram of experimental design; (B) Nucleocapsid formation was measured using NAGE assays and GFP expression was used to confirm transfection. (C) HBV transcripts and AID expression levels were determined using qRT-PCR. (D) AID dependent IgA switching was determined using flow cytometry. (E, F, G, H) CH12F3-2 cells were co-transfected with pPB and the indicated siRNA against mouse AID (simAID-1 and -2) or controls (siCtrl and siGFP), and after 6 hours incubation, cells were further stimulated with CIT for 3 days. HBV transcript levels, knock down efficiency of AID, and IgA switching were determined using qRT-PCR, western blotting, and flow cytometry, respectively. (I, J, K) A tetracycline promoter-regulating HBV plasmid (pTre-HBV) was stably transfected into CH12F3-2 transfectants expressing tetracycline-responsible transactivator (Tet-off). Established CH12F3-2 transfectants were designated CH12-HBV; (I) Schematic diagram of CH12-HBV; (J) CH12-HBV cells were incubated in the presence or absence of CD40 ligand, IL-4, or TGF-β1 (CIT) and tetracycline as indicated for 2 days to induce endogenous AID expression and IgA switching. HBV transcription and AID expression levels were determined using qRT-PCR. (K) IgA switching was detected according to surface expression of IgA using flow cytometry. (L) AICDA-deficient and-wild type BL2 cells were transfected with HBV plasmid (pPB), and qRT-PCR was performed at 3 day post-transfection. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (<i>t</i>-test). Data are representative of two to three independent experiments and error bars represent standard errors of the mean.</p

TGF-β1 upregulates APOBEC3 expression and suppresses HBV replication in Huh7 cells.

<p>Six hours after transfection of pPB, Huh7 cells were treated with TGF-β1 for 3 days, and HBV replication was evaluated. (A) qRT-PCR shows dose-dependent reduction of HBV transcripts by TGF-β1. (B) NC-DNA levels in secreted virions were also measured using qPCR. (C) Nucleocapsid NC-DNA and core protein levels in crude cytoplasmic extracts were assessed using NAGE assays. GAPDH protein levels in the same crude extracts were determined using western blotting. (D) Huh7 cells were treated with 150 ng/mL IL-4 or 10 ng/ml TGF-β1 for 3 days. Levels of HBV RNA and GAPDH mRNA were determined by Northern blot. Control: non-stimulated Huh7 cells. (E) Relative expression levels of APOBEC deaminases in non-stimulated Huh7 cells; Relative expression levels were determined using qPCR with cDNA from non-stimulated Huh7 cells and standard curves of control APOBEC deaminase DNA. Relative copy numbers of A3B were defined as one. (F) Induction of APOBEC deaminase expression in TGF-β1-treated Huh7 cells was estimated using qRT-PCR. Fold induction of APOBEC deaminases is shown in the top (10 ng/mL TGF-β1 for 24 or 48 h) and bottom (10 or 20 ng/mL TGF-β1 for 24 h) panels. (G) Huh7 cells were treated with indicated concentrations of TGF-β1 for 3 days. AID protein was immunoprecipitated using an anti-AID antibody (or an isotype control IgG, most right) and immunoprecipitated AID protein was determined by western blot. One lane contains immunoprecipitated protein harvested from 60% of 15 cm dish. All data are representative of two to four independent experiments. Error bars represent standard errors of the mean.</p

AID inducing HBV RNA reduction depends on Exosc3.

<p>(A, B) Huh7 cells were co-transfected with pPB and the indicated protein expression vectors, and were cultured for 3 days. Crude extracts (input) were then subjected to IP using an anti-FLAG antibody, and crude extracts and IP fractions were analyzed using western blotting. (C) Fold enrichment of HBV or HPRT transcripts upon anti-FLAG-Exosc3 IP; To determine RNA coprecipitation with the RNA exosome component Exosc3, Huh7 cells were transfected with pPB, pFLAG-Exosc3, and pCMV-AID (or pEGFP-C2), and were cultivated for 3 days. IP using anti-FLAG antibody was then performed, complexes of FLAG-Exosc3 were then eluted using free FLAG peptides, and the eluted RNA was analyzed using qRT-PCR. Combination of expression vectors used for transfection is the same with B (see numbers below the graph), and values in lane 3 were defined as 1. Error bars represent standard errors of the mean. (D) Associations of AID with RNA exosome proteins; Huh7 cells were co-transfected with indicated expression vectors, and were cultured for 3 days. Crude extracts (input) were subjected to IP with FLAG antibody, and crude extracts and IP fractions were analyzed using western blotting. Expression levels of GFP-Exosc7 were too low to be visualized in the crude extract (lanes 4 and 9, input), but GFP-Exosc7 was clearly detectable after FLAG-AID and FLAG-Exosc3 immunoprecipitation (lanes 4 and 9, IP). (E) Huh7 cells were co-transfected with pPB and either AID or GFP expression vectors and each of the siRNAs indicated in E and F, and cells were cultured for 3 days. HBV transcript levels, nucleocapsid formation, and Exosc3 expression were estimated using northern and western blotting, NAGE assays (E), and qRT-PCR analyses (F and G); siGFP and siCtrl were used as controls; **<i>P</i> < 0.01 (<i>t</i>-test); Data are representative of two to three independent experiments and error bars represent standard errors of the mean.</p

TGF-β1-mediated reduction of HBV transcripts depends on AID and Exosc3.

<p>Stable HBV transfectant Huh7 cells (7T7-8) were infected with recombinant lentiviruses to express indicated short hairpin (sh) RNA, and then cells were incubated in the presence or absence of 15 ng/ml TGF-β1 for 3 days. (A) Schematic diagram of experimental design; (B) AID expression levels in qRT-PCR and (C) IP western blotting. Crude extract before IP was also blotted (input). Crude extracts from TGF-β1-treated 7T7-8 transfectants were immunoprecipitated by anti-AID antibody. Loading control: anti-(adenosine deaminase acting on RNA) ADAR. (D) Exosc3 expression level in qRT-PCR or western blotting (E); shLuc was used as a control; (F) Reductions of HBV transcript levels following TGF-β1 treatment are compared between shAID-, shExosc3-, and shLuc-expressing 7T7-8 cells. HBV transcript levels of each non-stimulated transfectant are defined as 1; shLuc was used as a non-targeted control. *<i>P</i> < 0.05, **<i>P</i> < 0.01 (<i>t</i>-test), error bars represent standard errors of the mean. Data are representative of two to three independent experiments.</p

AID is responsible for TGF-β1-mediated reduction of HBV transcripts.

<p>To evaluate antiviral activity of indicated APOBEC proteins, Huh7 cells were co-transfected with FLAG-tagged A3A, A3C, A3F, A3G, GFP or GFP-tagged AID expression vectors and pPB. Cells were cultured for 3 days, and then HBV replication was estimated using NAGE assays (A). Protein expression is shown (A, bottom). qRT-PCR analyses of HBV transcripts (B), and qPCR analyses of NC-DNA in secreted virions (C). (D), Secreted virions in the culture medium and cytoplasmic extracts were treated with proteinase K and SDS to digest nucleocapsids, and levels of HBV DNA were determined using Southern blotting. (E), To evaluate contribution of indicated APOBEC proteins, Huh7 cells were co-transfected with pPB and the indicated siRNAs. Six hours later, cells were incubated in the presence or absence of 10 ng/mL TGF-β1. Three days later, total RNA was extracted, and qRT-PCR performed to determine expression levels of HBV transcripts, AID, A3A, and A3G. Although siAID significantly reduced AID expression and prevented the downregulation of HBV transcripts in TGF-β1-stimulated Huh7 cells (lane 1), siA3A and siA3G had no effects against the downregulation of HBV transcripts (lanes 2–4). siGFP was used as a control. Expression levels in lane 8 are defined as one fold induction. **<i>P</i> < 0.01 (<i>t</i>-test); Data are representative of two to three independent experiments and error bars represent standard errors of the mean.</p

Intact P protein is required for AID-mediated downregulation of HBV transcripts and AID associates with HBV P protein.

<p>(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.</p