Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

Xiuji Cui,Ji A Sohn,Rajeev Boregowda,Keith W Caldecott,Christoph Seeger,Felipe Cortes Ledesma,Jianming Hu,Rebecca Mcallister,Chiaho Shih

doi:10.1371/journal.pone.0128401

Abstract

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5’ end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5’ DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5’ DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5’ end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.

Highlights

An estimated 350 million persons worldwide are chronically infected with hepatitis B virus (HBV), 25% of whom will die from severe liver diseases including cirrhosis and hepatocellular carcinoma [1]
A recent study showed that pretreatment of the HBV and DHBV relaxed circular (RC) DNA–reverse transcriptase (RT) complex released from NCs with Tyrosyl DNA phosphodiesterase-2 (Tdp2) could make the 5’ end of the (-) strand susceptible to 5’ exonuclease digestion, suggesting that Tdp2 could release the RT protein from RC DNA [25]
Incubation with Tdp2 should cleave the tyrosyl-DNA bond and yield protein free minus strands. To assess this possibility experimentally, proteinase K-digested HBV RC DNA was treated with Tdp2, digested with the restriction endonuclease Sfc I and radioactively labeled with a Klenow fill-in reaction in the presence of [α-32P]-TTP

Summary

Introduction

An estimated 350 million persons worldwide are chronically infected with hepatitis B virus (HBV), 25% of whom will die from severe liver diseases including cirrhosis and hepatocellular carcinoma [1]. The genomic RC DNA is transported to the nucleus and converted (repaired) to an episomal covalently closed circular (CCC) DNA [5] that serves as the template for transcription of all viral RNAs. Unlike conventional retroviral reverse transcription, hepadnaviruses initiate minus or (-) strand DNA synthesis using a tyrosine (Y) residue within a unique terminal protein (TP) domain of the virally encoded reverse transcriptase (RT) itself as a primer (protein priming), a process that results in the covalent linkage of RT to the 5’ end of the (-) strand of RC DNA [6,7,8,9]. Upon completion of reverse transcription, mature RC DNA-containing nucleocapsids (NCs) are enveloped by viral surface proteins and secreted as virions or, alternatively, RC DNA is recycled back to the nucleus to amplify the pool of CCC DNA [5, 10, 11]

Methods

Results

Conclusion