Figure 1
Gradual reduction in the positive charge contents of HBc correlated with the gradual reduction in the size and amount of viral particle-associated HBV DNA and RNA.
(A) HBc consists of two distinct domains responsible for capsid assembly and viral RNA encapsidation. Three major phosphorylation sites at serine (S) 155, 162 and 170 are shown in blue34. Arginines (R) are shown in red. (B) The charge balance hypothesis (electrostatic homeostasis) highlights the electrostatic interactions between the positive charge from HBc arginine-rich domains (ARD) and the negative charge from encapsidated nucleic acids. Such a balanced or imbalanced electrostatic interaction in the capsid interior could regulate viral RNA encapsidation, DNA replication, capsid conformation, stability and assembly40,48,49. (C) Construction of 15 different ARD mutants with different R-to-A substitutions at different positions. Viral DNA and RNA from transfected culture were analyzed. (D) Upper panel: Both numbers and positions of positive-charged arginine could strongly influence the size and intensity of HBV DNA replicative intermediates by Southern blot analysis. RC: full-length relaxed circle DNA (4.0 kb). Heat-denatured HBV DNAs (100 °C, 5 min) banded as smearing signals starting from the 1.5 kb SS (single-strand) DNA position (lane 18). Smearing signals represent HBV DNA replicative intermediates with different MW. Lower panel: Intracellular capsid particles were assayed by native agarose gel electrophoresis and Western blot using an anti-HBc antibody. (E) The core particle-associated DNAs were analyzed by Whole Genome PCR (M&M10) and agarose gel electrophoresis with ethidium bromide staining. The mainstream population of encapsidated viral DNAs decreased in size when the arginine content was decreased. #Shorter form HBV DNA; *Full-length 3.2 kb HBV DNA (lane 17); FL-HBV: 3.2 kb full-length DNA amplified from an HBV plasmid (lane 19). (F) Upper panel: HBV core-associated viral RNAs were analyzed by Northern blot. Consistent with the gradual reduction in viral DNA sizes in (D,E), the size reduction of encapsidated RNA was associated with the gradual reduction of the positive charge content of HBc ARD. RI and Sp RNA: replicative intermediates of reverse-transcribing viral RNAs and spliced RNAs. Lower panel: total cytoplasmic HBV RNA and GAPDH were included as controls.
