The Majority of Duck Hepatitis B Virus Reverse Transcriptase in Cells Is Nonencapsidated and Is Bound to a Cytoplasmic Structure

Hepatitis B virus (HBV) replication requires reverse transcription of an RNA pregenome (pgRNA) by a multifunctional polymerase (HP). HP initiates viral DNA synthesis by using itself as a protein primer and an RNA signal on pgRNA, termed epsilon (H), as the obligatory template. We discovered a Mn 2؉ -dependent transferase activity of HP in vitro that was independent of H but also used HP as a protein primer. This protein-primed transferase activity was completely dependent on the HP polymerase active site. The DNA products of the transferase reaction were linked to HP via a phosphotyrosyl bond, and replacement of the Y63 residue of HP, the priming site for templated DNA synthesis, almost completely eliminated DNA synthesis by the transferase activity, suggesting that Y63 also serves as the predominant priming site for the transferase reaction. For this transferase activity, HP could use all four deoxynucleotide substrates, but TTP was clearly favored for extensive polymerization. The transferase activity was highly distributive, leading to the synthesis of DNA homo-and hetero-oligomeric and -polymeric ladders ranging from 1 nucleotide (nt) to >100 nt in length, with single-nt increments. As with H-templated DNA synthesis, the protein-primed transferase reaction was characterized by an initial stage that was resistant to the pyrophosphate analog phosphonoformic acid (PFA) followed by PFA-sensitive DNA synthesis, suggestive of an HP conformational change upon the synthesis of a nascent DNA oligomer. These findings have important implications for HBV replication, pathogenesis, and therapy. H epatitis B virus (HBV)is an important human pathogen and a member of the family Hepadnaviridae, which comprises a group of pararetroviruses replicating a double-stranded DNA genome through an RNA intermediate called pregenomic RNA (pgRNA) by a multifunctional viral polymerase protein (HP), a specialized reverse transcriptase (RT) (1-4). Structurally, HP is divided into four domains, including (from the N to the C terminus) the terminal protein (TP), spacer, RT, and RNase H domains (5-7). Although the RT and RNase H domains are conserved with those found in other RT enzymes, TP is unique to hepadnaviruses, and the spacer has no known function other than serving as a tether between the TP and RT domains (5, 7-11). As with other RTs, HP has RNA-and DNA-dependent DNA polymerase activity. Furthermore, HP also serves as a protein primer; specifically, a highly conserved Y residue in its TP domain (Y63) is used as a primer to initiate viral minus-strand DNA [(Ϫ)-DNA] synthesis (12, 13). This protein-primed reverse transcription requires a specific HBV RNA template called epsilon (Hε), located at the 5= end of the pgRNA, which is specifically recognized by HP and is thought to be an integral component of the HP holoenzyme (14-21). HP-Hε interactions trigger conformational changes in both the protein and RNA of the resulting ribonucleoprotein (RNP) complex, which are thought to be required functionally for HP to gain catalytic activity a...

Section: Figmentioning

confidence: 99%

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Jones

2013

“…To begin a structural assessment of DHBV P, we raised six immunoglobulin G monoclonal antibodies (MAbs) against the DHBV P terminal protein domain (amino acids 1 to 207) (43) and determined the exposure of their epitopes to in vitrotranslated P by immunoprecipitation. In vitro-translated P primes DNA synthesis in the presence of dGTP and ε (40) and hence is an excellent source of properly folded, enzymatically active P that is not buried within capsids.…”

Section: Resultsmentioning

confidence: 99%

Identification of an Essential Molecular Contact Point on the Duck Hepatitis B Virus Reverse Transcriptase

Cao¹,

Badtke²,

Metzger³

et al. 2005

Self Cite

The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription (reviewed in reference 9). It has a lipid envelope studded with viral glycoproteins that surrounds an icosahedral core particle composed of the core protein. Within the core particle are the viral nucleic acids and reverse transcriptase (P). Other hepadnaviruses infect woolly monkeys, woodchucks, ground squirrels, ducks, geese, and herons (6,17,29,31,32). Significant differences exist among the hepadnaviruses, but they share a high degree of hepatotropism, follow the same replication cycle, and have a nearly identical genetic organization.Hepadnaviral reverse transcription (34) occurs within cytoplasmic capsid particles. Reverse transcription begins with binding of P to an RNA stem-loop (ε) on the pregenomic RNA, and then this complex is encapsidated. Reverse transcription is primed by P itself, so minus-strand DNA is covalently linked to P (36, 40). Complexes containing P and the viral nucleic acids must be dynamic because three strand transfers are required to produce the mature circular viral DNA (21,22,38,41).The binding of P to ε requires the active participation of a molecular chaperone complex (13). In vitro reconstitution studies with recombinant duck hepatitis B virus (DHBV) P revealed that P-ε binding requires HSP90, HSP70, HSP40, HSP23, and HOP (2,11,14), although under certain circumstances only HSC70 and HSP40 are necessary (3). Because chaperones mod...

“…The polymerase inside the capsids cannot reach the substrate once part of the replicating RNA or DNA template is exposed irreversibly to the capsid exterior and becomes nuclease sensitive. It has also been reported that nonencapsidated DHBV polymerase is enzymatically inactive (56).…”

Section: Discussionmentioning

confidence: 99%

Exposure of RNA Templates and Encapsidation of Spliced Viral RNA Are Influenced by the Arginine-Rich Domain of Human Hepatitis B Virus Core Antigen (HBcAg 165-173)

Pogam

Chua

Newman

et al. 2005

179

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nucleaseresistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.The hepatitis B virus (HBV) core antigen (HBcAg) is a 22-kDa protein with multiple functions, including interactions with the pregenomic RNA (pgRNA) and the polymerase during encapsidation (10,31,41) and with the viral DNA during reverse transcription and DNA elongation (18), polymerization to form the nucleocapsid or core particles (4, 13), importing of HBV DNA to the nucleus (51), and targeting to the endoplasmic reticulum for envelope formation (5). The C-terminal region of HBcAg contains a protamine-like domain, which is rich in arginine and presumably binds to HBV RNA and DNA during pgRNA encapsidation and DNA replication (13,18,35). This arginine-rich domain has been shown to be dispensable for core particle assembly but not for viral replication (2, 4, 13, 30).