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

Abstract

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...

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation

“…A very recent report showed that mutations at another NTD site, V124, at the core-dimer interface (Fig. 7), close to I126, also led to a selective defect in mature RC DNA accumulation, although the defects of those mutants were attributed to RC DNA synthesis rather than destabilization (25). It is important to point out that the precise quantification of the synthesis versus degradation of the different DNA species was difficult, due to the dynamic nature of NC maturation and degradation, NC uncoating, RC DNA deproteination, and conversion to CCC DNA.…”

“…The role of the I126, together with that of the nearby V124 at the dimer interface (25) (Fig. 7), in mature NC formation/ integrity suggests that maturation-associated NC structural changes, perhaps triggered by the altered interactions of HBc with the dsDNA in mature NCs versus the ssDNA or pgRNA in immature NCs (52), may be initiated and/or propagated through the dimer interface.…”

“…The CTD is highly basic and dynamically phos-phorylated, which is thought to be important for viral RNA packaging and DNA synthesis (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The NTD has also been shown recently to play a role in viral DNA synthesis beyond its role in capsid assembly (25,26).…”

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly

“…Residue V124 of core protein forms part of the wall of the HAP pocket. Replacing V124 with amino acid residues with a hydrophobic side chain of different size partially fills the HAP pocket and alters capsid assembly and interaction with capsid assembly modulators (43,44). Specifically, the V124A mutation (Fig.…”

Hepatitis B Virus Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

“…Reverse transcription (RT) occurs within capsids. Cp is an active participant in genome replication, synthesizing first the minus-strand DNA and subsequently the dsDNA from the pgRNA template (12)(13)(14)(15). Three distinct template switches occur during genome replication, among which the second template switch determines if the major form of relaxed circular (RC) or the minor form of duplex linear (DL) dsDNA is synthesized (16).…”