Structure-guided functional analysis of the hepadnaviral polymerase - hepadnaviruses are partially double-stranded DNA viruses that replicate by protein-primed reverse transcription

The National Institute of Allergy and Infectious Diseases awarded Saint Louis University $627,222 on May 18, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to conduct structure-guided functional analysis of the hepadnaviral polymerase, with performance through April 30, 2031.

The research examines the four-domain viral polymerase responsible for hepadnaviral reverse transcription, with focus on hepatitis B virus (HBV), which causes approximately 1.1 million annual deaths. The work combines structural modeling with functional analysis of the polymerase's protein-priming, reverse transcriptase, and ribonuclease H activities. Specific aims include determining which polymerase sequences are essential for binding to the viral pregenomic RNA and for DNA priming, analyzing how the terminal protein domain cups over the catalytic core, and testing mechanistic predictions derived from the recently validated structural model of the polymerase. The project targets nucleos(t)ide analog drug development by clarifying the poorly understood enzymology of hepadnaviral protein-primed reverse transcription, which differs substantially from retroviral mechanisms despite HBV polymerase serving as a major drug target.

Work is performed in St. Louis, Missouri. The award is structured as a Project Grant, the standard assistance type for NIH research support to individual investigators and institutions.

This family includes duck hepatitis b virus (dhbv) with which hepadnaviral reverse transcription was discovered and human hepatitis b virus (HBV) that kills 1,100,000 people annually. HBV therapy primarily employs nucleos(t)IDE analog drugs that target the viral reverse transcriptase (RT) activity. Reverse transcription is catalyzed by the 4-domain viral polymerase (p) which has protein priming, RT, and ribonuclease h (rnase h) activities. The TP and spacer domains are unique to the hepadnaviruses. Reverse transcription starts with chaperone-mediated binding of p to the ε stem loop on the viral pregenomic RNA (pgrna). Reverse transcription is primed by a tyrosine in p's terminal protein domain (TP), templated by ε. The RT synthesizes the first strand of the viral DNA, and the rnase h destroys the pgrna to permit synthesis of the second DNA strand. P is a monomer, and the covalent linkage between p and the DNA persists throughout reverse transcription. Hepadnaviral protein-primed reverse transcription differs greatly from retroviral reverse transcription, but its enzymology is poorly understood even though HBV p is a major drug target. This knowledge GAP is in part due to the inability to determine the structure of p. We recently predicted the structure of p and validated the model. This revealed a novel fold in which the TP domain that primes reverse transcription is cupped over p's catalytic core of p, with the priming tyrosine on a loop over the RT active site. This model makes mechanistic predictions regarding reverse transcription and provides guidance for how to test the hypotheses. Premise: the molecular model of p enables in-depth mechanistic analyses of p structure, nucleic acid binding, and DNA priming by the enzyme for the first time. Aim 1. What p sequences are needed for ε binding and priming? We will define the minimal active form(s) of p for RNA binding and DNA priming, and identify residues of p that contact ε and are essential for priming. Aim 2. What are the structural alterations to p associated with the shift from the priming-incompetent to priming-competent state? We will define how the TP domain binds to the catalytic core of p, explore p's conformational shifts during priming, and determine how key RNA binding motifs are exposed during ε binding. Aim 3. How do conformational dynamics of p contribute to ε binding and priming? We will probe how p's flexibility affects ε binding and DNA priming using molecular dynamics plus pharmacological and mutational analyses. We will test effects of mutations affecting RNA binding and DNA priming on viral replication in cells. This study will fill major gaps in our understanding of hepadnaviral reverse transcriptase enzymology by defining the interactions holding p in its novel conformation, how p binds to ε, and how enzyme flexibility contributes to the early phases of reverse transcription. IT will also provide key information needed to develop non-active site inhibitors of HBV p to improve therapy for HBV patients.