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APOBEC3-induced mutation of the hepatitis virus B DNA genome occurs during its viral RNA reverse transcription into (-)-DNA.

ABSTRACT: APOBEC3s are innate single-stranded DNA cytidine-to-uridine deaminases that catalyze mutations in both pathogen and human genomes with significant roles in human disease. However, how APOBEC3s mutate a single-stranded DNA that is available momentarily during DNA transcription or replication in vivo remains relatively unknown. In this study, utilizing hepatitis B virus (HBV) viral mutations, we evaluated the mutational characteristics of individual APOBEC3s with reference to the HBV replication process through HBV whole single-strand (-)-DNA genome mutation analyses. We found that APOBEC3s induced C-to-T mutations from the HBV reverse transcription start site continuing through the whole (-)-DNA transcript to the termination site with variable efficiency, in an order of A3B >> A3G > A3H-II or A3C. A3B had a 3-fold higher mutation efficiency than A3H-II or A3C with up to 65% of all HBV genomic cytidines being converted into uridines in a single mutation event, consistent with the A3B localized hypermutation signature in cancer, namely, kataegis. On the other hand, A3C expression led to a 3-fold higher number of mutation-positive HBV genome clones, although each individual clone had a lower number of C-to-T mutations. Like A3B, A3C preferred both 5'-TC and 5'-CC sequences, but to a lesser degree. The APOBEC3-induced HBV mutations were predominantly detected in the HBV rcDNA but were not detectable in other intermediates including HBV cccDNA and pgRNA by primer extension of their PCR amplification products. These data demonstrate that APOBEC3-induced HBV genome mutations occur predominantly when the HBV RNA genome was reversely transcribed into (-)-DNA in the viral capsid.

SUBMITTER: Chen Z

PROVIDER: S-EPMC8321922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledHepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.ImportanceThe hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.

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APOBEC3-induced mutation of the hepatitis virus B DNA genome occurs during its viral RNA reverse transcription into (-)-DNA.

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