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. 2010 Jul;84(13):6387-99.
doi: 10.1128/JVI.00236-10. Epub 2010 Apr 21.

Inhibition of hepatitis B virus replication by MyD88 involves accelerated degradation of pregenomic RNA and nuclear retention of pre-S/S RNAs

Jianhua Li  1 , Shanshan Lin, Qiying Chen, Lu Peng, Jianwei Zhai, Yinghui Liu, Zhenghong Yuan
Affiliations

Inhibition of hepatitis B virus replication by MyD88 involves accelerated degradation of pregenomic RNA and nuclear retention of pre-S/S RNAs

Jianhua Li et al. J Virol. 2010 Jul.

Abstract

Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-alpha), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-alpha-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5'-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88.

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Figures

FIG. 1.
FIG. 1.
MyD88 inhibits HBV replication in HepG2.2.15 cells and in a mouse model. (A) HepG2.2.15 cells were mock infected or infected with Ad-EGFP or Ad-MyD88 or treated with 5,000 IU/ml IFN-α. The levels of viral RNA and core particle-associated DNA were determined by Northern (top) and Southern (middle) blot analyses, respectively, using a 32P-radiolabeled HBV DNA probe. After the detection of HBV RNA, the blots were stripped and rehybridized with a 32P-radiolabeled GAPDH DNA probe to control for gel loading. The positions of HBV pregenomic RNA (3.5 kb), pre-S1/S RNA (2.4 kb), and pre-S2/S RNA (2.1 kb) and the positions of relaxed circular (RC), single-stranded (SS) DNAs are indicated. MyD88 expression was confirmed by Western blot analysis using an anti-MyD88 antibody. The levels of the β-actin protein were determined to normalize for protein loading (bottom). (B) BALB/c mice were hydrodynamically coinjected with pHBV1.3 and pCMV/Myc or pCMV/Myc-MyD88. At 4 days postinjection, total liver DNA was extracted and subjected to digestion with HindIII restriction endonuclease. Input pHBV1.3 and viral core particle-associated DNA were examined by Southern blot analysis (top). The intensity of input pHBV1.3 in each lane was used as an internal control indicating equivalent transfection efficiency. Total liver RNA was analyzed by Northern blotting, and GAPDH was analyzed as a loading control (middle). MyD88 expression was confirmed by Western blot analysis using an anti-Myc antibody (bottom). Eight comparable pairs in which each part showed similar amounts of input DNA were obtained from two independent injections, and two representative pairs are shown. (C) Huh7 (top) and HepG2 (bottom) cells were cotransfected with the indicated amounts of pCIdA-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of HBV RNAs were determined by Northern blot analysis. GAPDH was analyzed as a loading control.
FIG. 2.
FIG. 2.
Knockdown of MyD88 expression by RNA interference partially abrogates IFN-α-induced inhibition of HBV replication. (A) Huh7 cells were transfected with 40 nM siRNAs _targeting EGFP (siEGFP) or MyD88. After 24 h, the cells were transfected with 20 nM the same siRNAs together with pHBV1.3 and a β-Gal control plasmid, followed by treatment with or without 5,000 IU/ml IFN-α for 48 h. Viral RNA and DNA were analyzed by Northern blotting and Southern blotting, respectively. (B) Viral RNA and DNA levels from three independent experiments were quantified by phosphorimaging and normalized against GAPDH levels and β-Gal activity, respectively. *, P < 0.05.
FIG. 3.
FIG. 3.
MyD88 downregulates HBV RNAs by a posttranscriptional mechanism. (A) Huh7 cells were cotransfected with 0.1 μg of each of four reporter plasmids for HBV promoters and enhancers (ENII/Cp, Sp1, Sp2, and ENI/Xp, respectively) and pCMV/Myc or pCMV/Myc-MyD88. The cells were harvested at 48 h posttransfection, and luciferase activity in the lysates was assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. (B). Huh7 cells were cotransfected with the indicated amounts of pCMV-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of the pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (C) Huh7 cells were cotransfected with the indicated amounts of pCDNA3.1-Luc and pCMV/Myc or pCMV/Myc-MyD88. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Vero cells were transfected as described above (B). Levels of pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.
FIG. 4.
FIG. 4.
MyD88 accelerates the decay of HBV pregenomic RNA in the cytoplasm. (A) Huh7 cells were cotransfected with pTet-HBV and pUHD-TA together with pCMV/Myc or pCMV/Myc-MyD88. At 39 h posttransfection, the cells were harvested directly upon the addition of doxycycline (Dox) or at 3, 6, or 9 h thereafter. The levels of pregenomic RNA were determined by Northern blot analysis. To normalize RNA loading, the same blots were hybridized with a GAPDH probe. (B) The half-life of the pregenomic RNA was determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed, and the levels of pregenomic RNA were normalized against RNA loading. (C) Huh7 cells were transfected as described above (A). Cytoplasmic (left) and nuclear (right) RNAs were prepared and analyzed by Northern blotting. (D and E) The half-lives of pregenomic RNA in the cytoplasm (D) and nucleus (E) were determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed and quantified by phosphorimaging, and the levels of pregenomic RNA were normalized against RNA loading. (F) Huh7 cells were transfected with 40 nM siRNA _targeting EGFP, DCP2, or EXOSC5. At 24 h posttransfection, the cells were transfected with 20 nM the same siRNA together with pHBV1.3 and pCMV/Myc-MyD88. At 48 h after the second transfection, the cells were harvested, and viral pregenomic RNA was analyzed by Northern blot analysis (top). The expression of _target proteins was evaluated by Western blot analysis using anti-DCP2 and anti-EXOSC5 antibodies (bottom).
FIG. 5.
FIG. 5.
MyD88-induced decay of viral pregenomic RNA was independent of the interaction of La with viral pregenomic RNA. (A) Huh7 cells were transfected with 1 μg of pCMV/Myc or pCMV/Myc-MyD88, with or without 0.5 μg of pHBV1.3, for 48 h. Western blot analysis for La was performed as described in Materials and Methods. (B) Huh7 cells were transfected with 1 μg of pCMV-HBV or pCMV-HBV M2 together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88. Levels of pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.
FIG. 6.
FIG. 6.
Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P < 0.05. (C) Huh7 cells were transfected with 1 μg of pCDNA3.1-Luc, Luc-HBV(1804-2454), or Luc-HBV(1151-1684) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Huh7 cells were transfected with 1 μg of Luc-ΔHBV(1151-1684) or Luc-ΔHBV(1804-2454) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (E) Huh7 cells were transfected with 1 μg of pCMV-HBV, pCMV-HBVΔ1804-2454, or pCMV-HBVΔ1151-1684 (also named pCMV-HBVΔPRE in this study) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.
FIG. 7.
FIG. 7.
MyD88 accelerates the decay of HBV pre-S2/S RNA in the nucleus. (A) Huh7 cells were transfected with 1 μg of pCDNA3.1-pre-S2/S or its deletion mutant of HBV [HBV(1151-1684)] together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of viral RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (B) Huh7 cells were cotransfected with pTRE2hyg-preS2/S and pUHD-TA together with pCMV/Myc or pCMV/Myc-MyD88. Cells were harvested directly upon the addition of doxycycline or 3, 6, and 9 h thereafter. (B) Nuclear (bottom) and cytoplasmic (top) RNAs were prepared and analyzed by Northern blotting. (C and D) The half-life of pre-S2/S RNA in the nucleus (C) and cytoplasm (D) was determined by Northern blot analysis and quantified by phosphorimaging. Data from two independent experiments performed in duplicate were analyzed and quantified by phosphorimaging, and the levels of pre-S2/S RNA were normalized against RNA loading.
FIG. 8.
FIG. 8.
The decay of HBV pre-S2/S RNA in the nucleus is associated with the deficiency in PRE-dependent nuclear transport. (A) Huh7 cells were transfected with 0.2 μg of pRSV-CAT or pRSV138PRE-CAT together with 0.4 μg of pCMV/Myc or pCMV/Myc-MyD88. At 48 h posttransfection, the cells were harvested, and CAT activity was measured. For each transfection, 0.1 μg of β-Gal was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P < 0.05. (B and C) Huh7 cells were transfected with the indicated amounts of pRSV138PRE-CAT and pCMV/Myc-MyD88 together with pCMV/Myc-NES(−)RanBP or pCMV/Myc-PTB1. At 48 h posttransfection, the cells were harvested, and CAT activity was measured as described above (A). (D and E) Huh7 cells were transfected with the indicated amounts of pRSV138PRE-CAT and pCMV/Myc-MyD88 together with pCMV/Myc-NES(-)RanBP or pCMV/Myc-PTB1 for 48 h. Cytoplasmic and nuclear RNAs were prepared and analyzed by Northern blotting with a 32P-radiolabeled CAT DNA probe. To normalize RNA loading, the same blots were hybridized with a GAPDH probe.
FIG. 9.
FIG. 9.
MyD88 transcriptionally inhibits the expression of PTB. (A) Huh7 cells were transfected with 1 μg of pCMV/Myc or pCMV/Myc-MyD88, with or without 0.5 μg of pHBV1.3, for 48 h. Western blot analysis for PTB and IκBα was performed as described in Materials and Methods. (B) Huh7 cells were transfected with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 together with 1 μg of pHBV1.3 for 48 h. Total RNA was prepared and analyzed by Northern blotting with a 32P-radiolabeled PTB DNA probe. (C) Huh7 cells were transfected as described above (B). At 44 h posttransfection, the cells were treated with actinomycin D. The cells were harvested directly upon the addition of actinomycin D or at 2 or 4 h thereafter. Total RNA was prepared, and PTB mRNA levels were determined by Northern blot analysis. (D) Two independent experiments performed in duplicate were analyzed, data were quantified by phosphorimaging, and the levels of PTB mRNA were normalized against RNA loading.
FIG. 10.
FIG. 10.
Proposed model for MyD88-mediated inhibition of HBV replication in hepatoma cells. See the text for a more detailed discussion. cccDNA, covalently closed circular DNA.
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