Figure 3. A charge-rebalance approach via reducing acidic residues of HBc.

(A) A cartoon illustrates the charge rebalance approach. (B) The dimeric structure of HBc (PDB ID:1QGT) with each monomer in green and orange ribbon modes. The side chains of four glutamic acids are located close to the capsid interior: E40 (cyan), E46 (yellow), E113 (white) and E117 (blue)¹⁸,⁷⁰,⁷¹,⁹⁰. (C) The schematic presentation of a 40-mer HBc in the context of a 240-mer icosahedral particle (T = 4) is illustrated using PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). Upper panel: E40 (cyan), E46 (yellow), E113 (white) and E117 (blue). Lower panel: all four acidic residues are in black. (D) Parental HBc mutant ARD-I + II + III + IV exhibited a very short DNA phenotype by Southern blot, which can be partially rescued by E-to-A single or double mutations at E46 or E113. (E) The Southern result in (D) can be confirmed by Whole Genome PCR analysis using core particle-associated viral DNAs as a template. (F) Upper panel: Northern blot analysis of HBc parental mutant ARD-I + II + III + IV and its charge rebalanced derivatives. Rescued viral RNAs with significantly increased size (between two dotted lines) were observed in HBc mutant E46A + E113A (last lane). Lower panels: Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (G) The Northern results in (F) was confirmed by RT-PCR analysis using core particle-associated viral RNAs. (H) Size distributions of encapsidated viral DNAs in WT, mutant ARD-I + II + III + IV, and its double-rescued mutant. Each clone was characterized by sequencing as described in Fig. 2D. The horizontal line represents the medium size of encapsidated viral DNA species. ***p < 0.001, Student’s t-test. (I) Novel spliced transcripts (1983 bp and 1959 bp) can be rescued in mutant ARD-I + II + III + IV containing E46A and E113A mutations. (J) Upper panel: The acidic residue E180 (green) is located in the ARD without a known structure¹⁸,⁵⁷. Lower panel: Defective DNA replication of mutant ARD-III + IV can be synergistically rescued by multiple E-to-A mutations in a dose-dependent manner using Southern blot analysis. (K) The encapsidated RNA of mutant ARD-III + IV can be efficiently rescued in a dose-dependent manner by multiple charge rebalance mutations using Northern blot analysis.