Figure 7
A cartoon summary of three major approaches used here in testing the HBV charge balance hypothesis.
(A) The short RNA phenotype in arginine-deficient HBc mutants (e.g., ARD-III + IV) can be rescued successfully by using a charge rebalance approach (Fig. 3A). Both E-to-A and S-to-A mutations can restore the efficient packaging of longer-sized viral RNA, suggesting that serine phosphorylation at the ARD could contribute negative charge to electrostatic homeostasis required in the capsid interior (Figs 3 and 4, and Supplementary Figure S3). Similarly, mutant ARD-I + II + III + IV contains the most severe arginine deficiency and encapsidates predominantly spliced mini-RNAs of the shortest size (Figs 1 and 2). (B) Hyper-phosphorylation of HBc is responsible for the biogenesis and maintenance of empty capsids (without encapsidated RNA) in both insect and human cell systems (Figs 5 and 6). Empty capsids can also be expressed in E. coli by engineering a mutant S7D bearing 7 phosphorylation-mimicking aspartic acids at HBc ARD (Fig. 5). (C) WT HBV with double phosphorylations at S162 and S170 of HBc is believed to preferentially encapsidated 3.5 kb pgRNA37,38,41. In contrast, mutant S3E, containing three S-to-E mutations at the major phosphorylation sites (S155E, S162E and S170E) preferentially encapsidated spliced viral RNA39. Mutant S3A contains three de-phosphorylation mimicking mutations at three major phosphorylation sites (S155A, S162A, and S170A), and is known to be deficient in viral RNA encapsidation37,38,41. Interestingly, in the replicon context in HuH-7 cells, mutants S3A, S5A, and S7A can package increasing amounts of cellular RNA (Fig. 6F). Furthermore, we predict here that when all three major sites are phosphorylated, the 2.2 kb spliced RNA (1959 nt in Fig. 2E) will be more fitting for encapsidation than the non-spliced 3.5 kb pgRNA. Grey: wild type serine; black: phosphorylated serine; orange: S-to-E mutations; green: S-to-A mutations.
