Abstract
Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature.
Highlights
Hepatitis B Virus (HBV) contains four major open reading frames (ORFs), which encode precore/core, surface, polymerase, and X proteins
The working hypothesis remains highly speculative, since there are many important issues that need to be addressed: (1) If charge balance is so important, how can it explain the formation of empty capsids without any encapsidated RNA in natural infection and cell culture42–46? (2) Does encapsidation of non-viral or cellular RNA, if any, play any role in the electrostatic interaction in HBV capsids? (3) As mentioned above, HBc arginine-rich domain (ARD) contains a large number of serine phosphorylation sites
Is it possible that phosphoserine could contribute to balance electrostatic interactions? If so, are they functionally interchangeable and substitutable to encapsidated RNA, DNA, or acidic residues of HBc? (4) In addition to a genetic approach via phospho-mimicking mutagenesis, can a more direct biochemistry approach be used in the studies of the potential involvement of phosphorylation in electrostatic homeostasis? (5) In our earlier experiments using a truncated mutant HBc contex[40,48], it remains unclear if the successful rescue in viral replication is due to the increased length of the cytoplasmic tail of HBc, or due to the increased arginine content in the ARD domain of HBc40? Can the charge rebalance experiment be performed in a more natural full-length context48?
Summary
HBV contains four major open reading frames (ORFs), which encode precore/core, surface (envelope), polymerase, and X proteins. By reducing negative charges at acidic residues or serine phosphorylation sites of HBc, we can restore fully or partially the shorter RNA and DNA phenotype of arginine-deficient ARD mutants.
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