Single hepatocytes show persistence and transcriptional inactivity of hepatitis B.

Ashwin Balagopal,Yasmeen S Saad,William O Osburn,Richard K Sterling,Tanner Grudda,Michael Murphy,Alan S Perelson,Ruy M Ribeiro,Kathleen Ward,Chloe L Thio,Mark S Sulkowski,Jeffrey Quinn,Hyon S Hwang,Yang Zhang

doi:10.1172/jci.insight.140584

Abstract

There is no cure for the more than 270 million people chronically infected with HBV. Nucleos(t)ide analogs (NUCs), the mainstay of anti-HBV treatment, block HBV reverse transcription. NUCs do not eliminate the intranuclear covalently closed circular DNA (cccDNA), from which viral RNAs, including pregenomic RNA (pgRNA), are transcribed. A key gap in designing a cure is understanding how NUCs affect HBV replication and transcription because serum markers yield an incomplete view of intrahepatic HBV. We applied single-cell laser capture microdissection and droplet digital PCR to paired liver biopsies collected from 5 HBV/HIV-coinfected persons who took NUCs over 2–4 years. From biopsy 1 to 2, proportions of HBV-infected hepatocytes declined with adherence to NUC treatment (P < 0.05); we extrapolated that eradication of HBV will take over 10 decades with NUCs in these participants. In individual hepatocytes, pgRNA levels diminished 28- to 73-fold during NUC treatment, corresponding with decreased tissue HBV core antigen staining (P < 0.01). In 4 out of 5 participants, hepatocytes with cccDNA but undetectable pgRNA (transcriptionally inactive) were present, and these were enriched in 3 participants during NUC treatment. Further work to unravel mechanisms of cccDNA transcriptional inactivation may lead to therapies that can achieve this in all hepatocytes, resulting in a functional cure.

Highlights

Chronic HBV is the leading cause of hepatocellular carcinoma and end-stage liver disease globally
Participants in this study were all HIV coinfected because the parent Hepatitis B Research Network (HBRN) study focused on HBV/HIV coinfection
Two individuals were not on HBV therapy at baseline but were on HIV therapy and had high plasma HBV DNA levels approximately 2 weeks before biopsy 1 (HB6 and HB2 had > 8 log10 IU/mL); 2 individuals were on dually active antiretroviral therapy (ART) containing nucleos(t)ide analogs (NUCs) with incomplete adherence and had intermediate HBV DNA levels (HB7 and HB3 had > 4 log10 IU/mL); and 1 individual was adherent to long-term NUC therapy with low levels of HBV DNA at biopsy 1 (HB4) (Table 1)

Summary

Introduction

Chronic HBV is the leading cause of hepatocellular carcinoma and end-stage liver disease globally. A major barrier to a cure is the covalently closed circular DNA (cccDNA) that resides and persists in the nucleus of every infected hepatocyte and that is the template for transcription of viral RNAs [2]. Despite advances in cell culture and animal models, we have few insights into human HBV and regulation of cccDNA transcription in the organ that it infects, the liver, especially in people receiving antiviral treatments. Upon infecting the human hepatocyte, rcDNA uncoats and is converted into cccDNA by host DNA polymerases in the nucleus. Thereafter, a suite of short and long viral mRNAs is transcribed by host RNA polymerase II These viral transcripts are exported into the cytoplasm and either translated into structural, regulatory, or replicative proteins, or packaged into new virions. The most commonly used current antivirals are nucleos(t)ide analogs (NUCs), often dually active against HIV and HBV, which inhibit the reverse transcriptase, thereby interrupting the conversion of pgRNA to rcDNA

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