Hepatitis B virus reverse transcriptase: diverse functions as classical and emerging targets for antiviral intervention

Abstract

Hepatitis B virus (HBV) infection remains a global health problem with over 350 million chronically infected, causing an increased risk of cirrhosis and hepatocellular carcinoma. Current antiviral chemotherapy for HBV infection include five nucleos(t)ide analog reverse transcriptase inhibitors (NRTIs) that all target one enzymatic activity, DNA strand elongation, of the HBV polymerase (HP), a specialized reverse transcriptase (RT). NRTIs are not curative and long-term treatment is associated with toxicity and the emergence of drug resistant viral mutations, which can also result in vaccine escape. Recent studies on the multiple functions of HP have provided important mechanistic insights into its diverse roles during different stages of viral replication, including interactions with viral pregenomic RNA, RNA packaging into nucleocapsids, protein priming, minus- and plus-strand viral DNA synthesis, RNase H-mediated degradation of viral RNA, as well as critical host interactions that regulate the multiple HP functions. These diverse functions provide ample opportunities to develop novel HP-targeted antiviral treatments that should contribute to curing chronic HBV infection.

Keywords: Hepadnavirus; RNA packaging; RNase H; hepatitis B virus; protein priming; reverse transcriptase; review.

Figure 1

HBV replication cycle. Upon entry into the cytosol, the viral capsid with its encapsidated viral genome, a partially DS, RC DNA covalently attached to HP, traffics toward the nucleus where the genomic DNA is repaired to form cccDNA. cccDNA is transcribed by host RNA polymerase II to produce all viral transcripts including the overlength pgRNA. pgRNA acts as the template for translation of HP (as well as the core or capsid protein). HP binds to an RNA structural element called epsilon (Hε) on the 5′ end of pgRNA. HP–Hε binding triggers pgRNA encapsidation followed by minus- and plus-strand DNA synthesis inside the capsid. The mature capsid, containing RC DNA, can then be enveloped and secreted or alternatively deliver RC DNA to the nucleus to amplify the cccDNA pool. cccDNA, covalently closed circular DNA; DS, double-stranded; RC, relaxed circular.

Figure 2

HP domain structure and host interactions. (A) HP is schematically depicted with its domains, important motifs and critical residues indicated. Motifs and residues are color-coded to denote the known steps of HBV replication for which they are important, as outlined in the box in the lower right corner. Small boxes A–G denote the conserved regions across reverse transcriptases. Minimal regions of HP required for RNA binding are represented as green boxes. *, verified function in DHBV but not HBV. ** denotes the fact that the YMDD polymerase active site is required for both protein priming and all subsequent DNA synthesis. (B) Reported antiviral and proviral HP-binding factors. eIF4E is listed with a “?” because it is anticipated, but not yet verified, to promote viral replication. YMDD, tyrosine–methionine–aspartate–aspartate.

Figure 3

Hε requirements for HP interaction. (A) Hε is depicted with the conserved lower stem, internal bulge, upper stem and apical loop elements. Additionally, residues involved in priming are indicated in the internal bulge region. (B) Hε residues important for interaction with HP (blue oval) are indicated.

Figure 4

HP structural changes during protein priming and DNA elongation. (A) The initiation stage of protein priming. The HP–Hε complex initiates protein priming by adding a single dGMP residue to Y63 of the TP domain using the YMDD polymerase active site. (B) The DNA polymerization stage of protein priming. Following the initiation of protein priming, HP adds two dAMP residues to the dGMP that is already attached to TP. Both stages of protein priming use the internal bulge of Hε as the specific obligatory template. (C) DNA elongation following protein priming. The HP–dGAA covalent complex is transferred from Hε to a complementary sequence at the 3′ end of pgRNA where minus-strand DNA elongation continues. During each stage, distinct HP conformations are thought to be required for HP to carry out the different steps of viral DNA synthesis, as depicted by changes in the domain shape and color. The T3 motif is represented as a blue oval located in the TP domain; the RT1 motif is represented as an orange rectangle located in the RT domain. YMDD, tyrosine–methionine–aspartate–aspartate.

Figure 5

Hε requirements for HP–Hε formation, protein priming and RNA packaging. HP–Hε RNP formation requires the Hε bulge, but the apical loop and the distance between the 5′ cap and Hε are not critical (non-critical elements are designated as light gray, while the critical elements are highlighted with red). On the other hand, protein priming and RNA packaging require both the Hε internal bulge and apical loop, as well as a short distance between the 5′ cap and Hε. Modified from J Virol 2012; 86(9): 5134–5150. doi: 10.1128/JVI.07137-11, copyright © 2012, with permission from American Society for Microbiology.

Figure 6

Antiviral compounds targeting polymerase-ε binding and protein priming. DP–Dε binding (A) and HP priming (B) are depicted along with known corresponding inhibitors. * denotes that porphyrins can also inhibit HP–Hε binding. CLV, clevudine; ETV, entecavir; TFV, tenofovir.