Figure 4
A charge rebalance approach via reducing serine phosphorylation at HBc ARD.
(A) HBc ARD contains 7 potential serine phosphorylation sites (blue). (B) The replication defect of mutant ARD-III + IV can be partially rescued via a single S-to-A mutation at S155 and S170 by Southern blot analysis. (C) The short RNA phenotype of mutant ARD-III + IV can be significantly rescued via a single S-to-A mutation at S155, S162, S170 by Northern blot analysis. Mock: untransfected HuH-7 cells. Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (D) The replication defect of mutant ARD-III can be partially rescued by a charge rebalance mutation S162A by Southern blot analysis. Combining mutations S162A and ARD-III efficiently restored the full-length SS DNA, albeit no full-length RC DNA (see Discussion). (E) The defect in viral RNA packaging of mutant ARD-III can be efficiently rescued by a second mutation S162A by Northern blot analysis. Total cytoplasmic HBV RNA and GAPDH RNA were included as controls. (F) The Northern result in (E) can be confirmed by RT-PCR analysis. (G) The replication defect of mutant ARD-III + IV can be rescued, in the increasing order of efficiency, via single mutations S181A, S168A, S176A, and S178A at minor phosphorylation sites, by Southern blot analysis. (H) The defective DNA replication of HBc mutant ARD-III + IV can be rescued in a dose-dependent manner by combinations of multiple rebalance mutations S155A, S176A, S181A, and E180A. In triple-rescue mutants, clones #1 and #3 are two independent E. coli isolates. All the lanes shown here are from the same gel. (I) The RNA encapsidation defect of HBc mutant ARD-III + IV can be rescued in a dose-dependent manner via combinations of multiple rebalance mutations S155A, S176A, S181A, and E180A, by Northern blot analysis. HBV total cytoplasmic RNA and GAPDH were included as controls. All the lanes shown here are from the same gel.
