Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription

Abstract

Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls, making the ssDNA accessible to deaminase activity.

Distribution and difference of APOBEC‐induced mutations in the TpCpW context of HBV DNA between HCC and non‐HCC

“…However, in its immediately adjacent 1000 to 2000 nts region, the r apo value was the lowest (III area r apo = 1.19; IV area r apo = 1.09), and the subsequent r apo values showed a gradual increase (V region r apo = 1.46; VI region r apo = 2.12). The phenomenon of higher APOBEC‐induced mutation distribution in the minus strand of HBV DNA 1 to 1000 nts region was consistent with the results of previous studies . Nair et al suggested that high APOBEC‐induced mutations in this region might occur during rcDNA synthesis in the viral capsid.…”

“…APOBEC‐induced mutations have been found in the HBV DNA sequence and were mainly found in the HBV DNA minus strand . However, these findings mainly emanated from the cell model overexpressing APOBECs and a number of cases of serum and liver cirrhosis tissue samples .…”

Advances in Targeting the Innate and Adaptive Immune Systems to Cure Chronic Hepatitis B Virus Infection

“…It was firstly reported in 2005 that human APOBEC3 enzymes are able to extensively edit HBV DNA strands via cytidine deamination (77). In HBV-harboring HepAD38 and HepG2.2.15 cells, cytidine deaminases found endogenously could edit 10-25% of the HBV rcDNA genome within the viral capsid (78). Meanwhile, Hsp90 enhances APOBEC-3-mediated DNA deamination activity in HBV (79).…”

Biological basis for functional cure of chronic hepatitis B

“…This discrepancy could be explained by the sensitivity of the different G‐A detection methods used. Indeed, the asymmetric deamination pattern of the HBV genome suggested that it was edited by cytidine deaminases during reverse transcription, inside viral nucleocapsids . In this study, cells were not subjected to cytokine treatment to activate APOBEC proteins, and even in these conditions, deamination by endogenously active APOBEC was evident.…”

“…Indeed, the asymmetric deamination pattern of the HBV genome suggested that it was edited by cytidine deaminases during reverse transcription, inside viral nucleocapsids. 35 In this study, cells were not subjected to cytokine treatment to activate APOBEC proteins, and even in these conditions, deamination by endogenously active APOBEC was evident. Since APOBEC proteins could also inhibit HBV devoid of its deaminase activity, it was hypothesized that this was due to inhibition of minus strand synthesis or pgRNA packaging.…”