Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

Abstract

Hepatitis B virus (HBV) chronically infects >250 million people. It replicates by a unique protein‐primed reverse transcription mechanism, and the primary anti‐HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains compared to only two in most reverse transcriptases: the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviruses, HBV P has resisted structural analysis for decades. Here, we exploited computational advances to model P. The TP wrapped around the RT domain rather than forming the anticipated globular domain, with the priming tyrosine poised over the RT active site. The orientation of the RT and RNase H domains resembled that of the retroviral enzymes despite the lack of sequences analogous to the retroviral linker region. The model was validated by mapping residues with known surface exposures, docking nucleic acids, mechanistically interpreting mutations with strong phenotypes, and docking inhibitors into the RT and RNase H active sites. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses infecting rodent to fish hosts and a nackednavirus, but not in other non‐retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will advance mechanistic analyses into the poorly understood enzymology of HBV reverse transcription and will enable drug development against non‐active site targets for the first time.

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target

“…It is important to note that multiple retroviral-derived homology models of reverse transcriptase (RT) domains exist for HBV [ 59 , 60 , 61 ]. In addition, ab initio predicted models have been recently reported for the terminal protein (TP) domain [ 62 ] and for the entire P protein [ 63 ]. While these predicted structures [ 59 , 60 , 61 , 62 , 63 ] may provide valuable platforms for future drug discovery and design, experimentally derived structures are preferred.…”

Small Molecule Drugs Targeting Viral Polymerases

“…At present, no experimental structural data are available for this polymerase. However, a combination of in vitro studies, such as mutagenesis experiments, and in silico investigations, including sequence comparisons, homology modeling, and molecular dynamics simulations [83], led to the generation of 3D protein models [84][85][86]. Indeed, as HBV and HIV polymerases have a certain degree of sequence identity (~35%), several research teams have undertaken modeling of the HBV enzyme using the known 3D structure of the HIV protein as a template [83,[87][88][89].…”

“…Indeed, as HBV and HIV polymerases have a certain degree of sequence identity (~35%), several research teams have undertaken modeling of the HBV enzyme using the known 3D structure of the HIV protein as a template [83,[87][88][89]. The most recent 3D protein model of the HBV RT domain was developed using Alphafold program, providing structural insights into this challenging enzyme [86]. Specifically, the RT domain model distinctly revealed the catalytic Asp residues-Asp83, Asp205, and Asp206-responsible for chelating the two crucial Mg 2+ ions necessary for DNA synthesis, mirroring their positions in the HIV RT (i.e., Asp110, Asp185, and Asp186) [86].…”

“…The most recent 3D protein model of the HBV RT domain was developed using Alphafold program, providing structural insights into this challenging enzyme [86]. Specifically, the RT domain model distinctly revealed the catalytic Asp residues-Asp83, Asp205, and Asp206-responsible for chelating the two crucial Mg 2+ ions necessary for DNA synthesis, mirroring their positions in the HIV RT (i.e., Asp110, Asp185, and Asp186) [86].…”

Targeting Ribonucleases with Small Molecules and Bifunctional Molecules

“…Several molecules, such as the pyrimidinol carboxylic acid 11 that showed an IC 50 of 0.18 μM against HIV RNase H domain did not inhibit viral replication in HIV infectivity assays . Complex structures of HIV RNase H domain and small molecules have been solved and clearly showed the binding mode involving the three-oxygen pharmacophore interacting with the metal ions. ,− The RNase H domain active site is a shallow pocket difficult to be drugged without metal chelation . Active-site analysis revealed homology to the catalytic center of HIV integrase that contains two divalent metal ions and is responsible for inserting viral DNA into host genomic DNA .…”