Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Charles M. Rice
Abstract
Hepatitis B virus (HBV) is a partially double-stranded DNA virus that replicates through reverse transcription. Transcription from the HBV core promoter generates two genome-length RNAs: pregenomic RNA (pgRNA) and precore RNA (pcRNA). pcRNA contains the entire pgRNA sequence plus an additional 32 nucleotides at its 5’ end that include the precore start codon, enabling translation of the precore protein, the precursor of the secreted HBV e antigen (HBeAg). Whereas pgRNA is translated to produce core and polymerase and serves as the template for reverse transcription by cis-acting HBV polymerase, pcRNA has long been considered unable to support HBV DNA synthesis. Although precore is dispensable for viral replication, it is important for chronic HBV infection. Clinically, the G1896A mutation, which introduces a premature stop codon that abolishes precore translation, is highly prevalent among HBeAg-negative patients and is strongly associated with progressive liver disease. Despite this clinical importance, the biology and functions of HBV pcRNA remain incompletely understood, in part because conventional plasmid-based systems cannot readily distinguish pgRNA and pcRNA and generate high background DNA signals that confound detailed mechanistic studies. Here, I adapted an HBV RNA-based transfection system to systematically interrogate wild-type pgRNA and pcRNA, engineered pcRNA variants that abolish precore translation, and the naturally occurring G1896A mutant pcRNA. While wild-type pcRNA supported inefficient polymerase production and minimal HBV DNA synthesis, precore-translation-defective mutants and the G1896A mutant showed efficient polymerase production and HBV DNA production. Notably, polymerase translated from pcRNA and its variants preferentially acted in trans to reverse transcribe other ε-containing RNAs, in contrast to polymerase translated from pgRNA, which functioned predominantly in cis. These findings identify pcRNA as an underappreciated source of functional HBV polymerase with trans-acting activity. I next examined the implications of this mechanism for HBV pgRNA splice variants. HBV splicing variants have garnered increasing attention due to their association with liver disease progression in chronic HBV-infected patients. The most abundant splicing variant, SP1, does not support functional polymerase expression, yet SP1-derived DNA is detected in patient sera, implying that polymerase supplied in trans may drive SP1 DNA synthesis. Using co transfection assays, I showed that pcRNA-derived polymerases, particularly those from the G1896A mutant, efficiently reverse transcribe SP1 RNA in trans, yielding increased SP1-derived DNA and providing a mechanistic explanation for the biogenesis of SP1-derived DNA. Finally, I found that the pcRNA mutant terminating precore translation within the e proximal region exhibited impaired DNA production yet enhanced trans activity, consistent with a model in which a terminating ribosome occludes the 5’ e and limits polymerase binding in cis. To further define how precore translation impairs pcRNA reverse transcription, I performed a systematic scan of the pcRNA library containing targeted start and stop mutations across the precore region. This analysis allows mapping translation-dependent ε disruption at amino-acid position resolution. The data supports the model that translation of the precore ORF disrupts the 5’ ε RNA element required for polymerase binding, packaging, and initiation of reverse transcription. Altogether, this work demonstrates that pcRNA produces catalytically competent HBV polymerase, but that ongoing precore translation disrupts the 5’ e element and prevents HBV polymerase binding in cis, thereby biasing pcRNA-derived polymerase toward trans-acting reverse transcription of non-canonical 5’ ε-containing RNAs. These findings provide a mechanistic basis for the biogenesis of SP1-derived DNA and provide new insights into how translation-coupled remodeling of ε shapes HBV RNA selection for packaging and reverse transcription.
License and Reuse Information
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Recommended Citation
Zhang, Mengyin, "Noncanonical Roles of Hepatitis B Virus Precore RNA in Reverse" (2026). Student Theses and Dissertations. 868.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/868
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy