In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection
Xiaonan Zhang, … , Zhanqing Zhang, Zhenghong Yuan
Xiaonan Zhang, … , Zhanqing Zhang, Zhenghong Yuan
Published February 22, 2016
Citation Information: J Clin Invest. 2016;126(3):1079-1092. https://doi.org/10.1172/JCI83339.
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In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Abstract

Persistent hepatitis B virus (HBV) infection is established by the formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver. Very little is known about the intrahepatic distribution of HBV cccDNA in infected patients, particularly at the single-cell level. Here, we established a highly sensitive and specific ISH assay for the detection of HBV RNA, DNA, and cccDNA. The specificity of our cccDNA probe set was confirmed by its strict intranuclear signal and by a series of Southern blot analyses. Use of our in situ assay in conjunction with IHC or immunofluorescence uncovered a surprisingly mosaic distribution of viral antigens and nucleic acids. Most strikingly, a mutually exclusive pattern was found between HBV surface antigen–positive (HBsA-positive) and HBV DNA– and cccDNA-positive cells. A longitudinal observation of patients over a 1-year period of adeforvir therapy confirmed the persistence of a nuclear reservoir of viral DNA, although cytoplasmic DNA was effectively depleted in these individuals. In conclusion, our method for detecting viral nucleic acids, including cccDNA, with single-cell resolution provides a means for monitoring intrahepatic virological events in chronic HBV infection. More important, our observations unravel the complexity of the HBV life cycle in vivo.

Authors

Xiaonan Zhang, Wei Lu, Ye Zheng, Weixia Wang, Lu Bai, Liang Chen, Yanling Feng, Zhanqing Zhang, Zhenghong Yuan

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Figure 3

Southern blot analysis of protein-free and total DNA from liver biopsies.

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Southern blot analysis of protein-free and total DNA from liver biopsies...
(A) Protein-free DNA from liver biopsies (~5 mg) and HepAD38 cells (1 × 107 cells) was extracted using a modified Hirt’s procedure. DNA was left untreated or digested with T5 exonuclease or SpeI restriction nuclease, followed by Southern blotting using riboprobe 1. (B) Protein-free DNA from a liver biopsy and HepAD38, together with core particle DNA from a biopsy was left untreated (lanes 2, 6, and 11) or heated with 75°C (lanes 3, 7, and 12), 85°C (lanes 4, 8, and 13), or 95°C (lanes 5, 9, and 14) for 5 minutes and subjected to Southern blot analysis using riboprobe 1. Temp., temperature. (C) Protein-free DNA from a liver biopsy and HepAD38 cells was left untreated or treated with topoisomerase I (Topo I) and subjected to Southern blot analysis using riboprobe 1. M, molecular marker (2.0 and 3.2 kb). (D) Total DNA (lanes 2–7 and 11–16) and core particle DNA (lanes 8 and 9 and 17 and 18) from liver biopsies and HepAD38 cells was predigested with RNase A, RNase H alone (lanes 2, 5, 8, 9, 11, 14, 17, and 18), or in combination with PSD (lanes 3, 6, 12, and 15) or T5 exonuclease (lanes 4, 7, 13, and 16) for 1 hour at 37°C. The treated DNA was hybridized with minus strand–specific riboprobe 1 (lanes 1–9) or gap region–specific riboprobe 2 (lanes 10–18). Images in B–D (right panels) were generated by joining noncontiguous lanes on the same blot.