The Sequence of the RNA Primer and the DNA Template Influence the Initiation of Plus-strand DNA Synthesis in Hepatitis B Virus

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101

Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. IMPORTANCE The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target.The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment. Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide and contributes to 780,000 deaths per year (1). Currently there is no reliable cure for chronic hepatitis (2, 3). HBV is an enveloped double-stranded DNA (dsDNA) virus with an icosahedral core. HBV replicates its genome through an RNA intermediate. During replication, the capsid protein (Cp; also called the core protein) assembles around pregenomic RNA (pgRNA) and reverse transcriptase (the P protein) to form RNAfilled cores (4-8). Unlike HIV, reverse transcription of the HBV genome is a prerequisite for the progeny viruses to leave the host cell (5, 9).HBV reverse transcription is a complex reaction involving three template switches. P protein is incorporated into capsids durin...

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confidence: 99%

The Interface between Hepatitis B Virus Capsid Proteins Affects Self-Assembly, Pregenomic RNA Packaging, and Reverse Transcription

Tan

Pionek

Unchwaniwala

et al. 2015

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101

“…1C). As minus-strand DNA is synthesized, the RNase H activity of the P protein digests the pgRNA but leaves the 5Ј ϳ17 nucleotides intact (11,25,66). In a majority of cases, this RNA fragment undergoes a template switch from the 3Ј end of the minus strand to anneal to a complementary 11-nucleotide sequence near the 5Ј end, termed DR2, and primes plus-strand DNA synthesis at DR2 (primer translocation; Fig.…”

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confidence: 99%

The Arginine Clusters of the Carboxy-Terminal Domain of the Core Protein of Hepatitis B Virus Make Pleiotropic Contributions to Genome Replication

Lewellyn

Loeb

2011

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The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.Hepatitis B virus (HBV) replicates its genome via reverse transcription of a pregenomic RNA (pgRNA). This process ultimately produces a double-stranded DNA with a relaxedcircular conformation (rcDNA) (for a review, see reference 57). A distinguishing feature of HBV reverse transcription is that it occurs within the viral capsid. In the absence of HBV capsids, the HBV polymerase (P) is not able to produce rcDNA genomes from pgRNAs (36,58,61). Observations like these suggest that the capsid participates in reverse transcription.HBV capsids are icosahedra with primarily Tϭ4 symmetry (9,16,19). Each capsid contains 120 dimers of the core protein (16,69), and capsids can assemble in the absence of other viral components (59). A region of the core protein that is a candidate for a role in reverse transcription is the carboxy-terminal region of 34 amino acids, known as the carboxy-terminal domain (CTD). Capsids without the CTD assemble normally but do not support genome replication (8,47,70). The CTD can be attached to the capsid interior at the 5-or quasi-6-fold axes (71). Also, the CTD can bind to nucleic acids in vitro (28). Nearly 50% of the residues in the CTD are arginines, which give the CTD a net positive charge. Fourteen of the sixteen arginines are grouped into four clusters of three or four (see Fig. 2A for the amino acid sequence). This arrangement is conserved among mammalian hepadnaviruses ( Fig. 2A), suggesting it is important for function. Serines at positions 155, 162, and 170 are also conserved and can be phosphorylated in HBV (17,21,40). However, it is not fully understood which features of the CTD (arginine clusters, serine phosphoacceptor sites, or other) contribute to genome replication. The goal of the present study is to understand how each arginine cluster contributes to genome replicat...

“…To determine a step at which these mutants are defective, primer extension analysis with the 1744-R primer was carried out to measure the amount of plus-strand DNA synthesis initiated at DR2 (Fig. 6A), as previously performed (26). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis

Shin

Park

et al. 2014

Hepatitis B virus (HBV) synthesizes its DNA genome through reverse transcription, which is catalyzed by viral polymerase (Pol). Previous studies suggested that the RNase H domain of hepadnaviral Pol may contribute to multiple steps of the viral genome replication, such as RNA encapsidation and viral DNA synthesis. However, specific residues of the RNase H domain that contribute to viral reverse transcription have not been determined. Therefore, we employed charged-to-alanine scanning mutagenesis to generate a set of singlesubstitution mutants of the RNase H domain and then analyzed their ability to support viral reverse transcription. Southern blot analysis showed that three mutants (R703A, D777A, and R781A mutants) yielded significantly reduced amounts of viral DNAs. However, none of these mutants were defective in RNA encapsidation. The data indicated that in the R703A and D777A mutants, minus-strand DNA synthesis was incomplete due to loss of catalytic activity of RNase H. In contrast, in the R781A mutant, the minus-strand DNA synthesis was near complete to some extent, while the plus-strand DNA synthesis (i.e., relaxed circular DNA) was severely impaired due to the defect in RNase H activity. Overall, our analysis revealed that three charged residues of the HBV Pol RNase H domain contribute to the catalysis of RNase H in removing the RNA template, but not in the RNA encapsidation.