Testing the balanced electrostatic interaction hypothesis of hepatitis B virus DNA synthesis by using an in vivo charge rebalance approach

Abstract

Previously, a charge balance hypothesis was proposed to explain hepatitis B virus (HBV) capsid stability, assembly, RNA encapsidation, and DNA replication. This hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ARD) of the core protein (HBc) and the negative charge from the encapsidated nucleic acid. It remains unclear if any of the negative charge involved in this electrostatic interaction could come from the HBc protein per se, in addition to the encapsidated nucleic acid. HBc ARD IV mutant 173GG and ARD II mutant 173RR/R157A/R158A are arginine deficient and replication defective. Not surprisingly, the replication defect of ARD IV mutant 173GG can be rescued by restoring positively charged amino acids at the adjacent positions 174 and 175. However, most interestingly, it can be at least partially rescued by reducing negatively charged residues in the assembly domain, such as by glutamic acid-to-alanine (E-to-A) substitutions at position 46 or 117 and to a much lesser extent at position 113. Similar results were obtained for ARD II mutant 173RR/R157A/R158A. These amino acids are located on the inner surfaces of HBc icosahedral particles, and their acidic side chains point toward the capsid interior. For HBV DNA synthesis, the relative amount of positive versus negative charge in the electrostatic interactions is more important than the absolute amount of positive or negative charge. These results support the concept that balanced electrostatic interaction is important during the viral life cycle.

FIG. 1.

Arginine-deficient HBc mutants SVC173GG and SVC173/R157A/R158A exhibited a short DNA phenotype by complementation and Southern blot analysis. (A) To test the balanced electrostatic-interaction hypothesis, we engineered various HBc mutants with different arginine contents. The experimental approach is illustrated in the cartoon. (B) Amino acid sequence comparisons among the WT and HBc mutants. The hyphens represent amino acid sequences identical to that of the parental WT HBc. Roman numbers I to IV indicate the four different ARDs at the C terminus of HBc. The names SVC 173RR and SVC173 are used interchangeably in this paper. ARD IV mutant 173GG contains two arginine-to-glycine substitutions at positions 172 and 173, while ARD II mutant SVC173/R157A/R158A contains two arginine-to-alanine substitutions at positions 157 and 158. To further test the balanced electrostatic-interaction hypothesis, we restored arginines at the new positions 174 and 175 in mutant 175GGRR. (C, top) Plasmid 1903, an HBV genomic dimer containing an ablated core AUG initiation codon, was cotransfected with WT or various mutant core expression plasmids into Huh7 cells. HBV core-associated DNAs were analyzed by Southern blot analysis. The positive control mutant SVC173RR was WT-like in viral DNA synthesis (17, 20). More mature RC DNA of HBV was almost undetectable in mutants 173GG and 173/R157A/R158A, even after very long exposure to X-ray film. The replication defect of mutant 173GG could be rescued in mutant 175GGRR. The asterisk highlights the defect in synthesizing full-length viral DNA. In contrast, the black dots in lane SVC175GGRR highlight the functional rescue of full-length HBV DNA synthesis. SS, ssDNA replicative intermediate. (Bottom) Capsids collected from transfected cell lysates were measured by Western blot analysis using rabbit anti-core antibody.

FIG. 2.

Experimental design of a charge rebalance approach by converting acidic residues at specific positions in the assembly domain of HBc into neutral amino acids. (A, top) Stereo view of the HBV capsid protein dimer (PDB 1QGT) (6, 7, 36). Four residues, E40, E46, E113, and E117, are shown as yellow, pink, green, and cyan sticks, respectively. Unlike that of E40, the side chains of glutamic acids at E46, E113, and E117 point toward the capsid interior. E40 is farther away from the capsid interior than E46, E113, and E117, as is more clearly shown in the illustration of a hexamer (bottom). This dimer structure was created with Discovery Studio 2.0, and the hexamer structure was generated by using Insight II (Accelrys Software Inc.). (B) Sequence alignment of primate and rodent hepadnavirus core protein amino acids 36 to 49 and 110 to 122, with amino acids 40, 46, 113, and 117 in boldface. Acidic residues at 40, 46, and 117 are evolutionarily conserved. (C) To take a new approach to testing the balanced electrostatic-interaction hypothesis, we engineered E-to-A and E-to-R core mutants in the context of an arginine-deficient mutant, 173GG.

FIG. 3.

The replication defect of arginine-deficient core mutant SVC173GG can be rescued by reducing the negative charge in the assembly domain via E46A, E113A, or E117A mutations. (A) Mutations E40A, E40R, E46A, and E46R were introduced into the ARD IV mutant SVC173GG. (B) These E40 and E46 core mutants were tested for the ability to rescue plasmid 1903 by cotransfection and Southern blotting, as described in the legend to Fig. 1C. Unlike mutants E40A, E40R, and E46R, mutant E46A, in the context of 173GG, can result in a WT-like DNA phenotype. The black dots highlight the successful rescue of the replication defect of mutant 173GG. (Bottom) All mutant capsids in cell lysates were shown to be stable by Western blotting. (C) Mutations E113A and E117A were introduced into core mutant SVC173GG. (D) Better efficiency of rescue was observed in mutant E117A. The black dots highlight the rescue of more mature RC form DNA synthesis. (Bottom) Mutant capsids collected from cell lysates were as stable as wild-type capsids by Western blot analysis. (E) Heat denaturation experiments revealed that all three rescued mutants can display strong signals at the full-length ssDNA position; however, the signal of full-length RC DNA of mutant 173GG/E113A is relatively very weak. The parental mutant 173GG exhibited weak signals at the ssDNA position and no detectable signal at the full-length RC position. M, size marker.

FIG. 4.

Like that of the ARD IV mutant 173GG (Fig. 3), the replication activity of ARD II mutant SVC173/R157A/R158A can be enhanced by the single mutations E46A, E113A, and E117A. The experimental design of transfection and Southern blot analysis was similar to that described in the legends to Fig. 1 and 3. (A) Combinations of these mutations did not further enhance the HBV DNA signal. On the contrary, triple rescue with E46A, E113A, and E117A reduced the replication activity in at least three independent experiments. (B) Heat denaturation experiments revealed that, despite the absence of full-length RC DNA signal, a weak signal of full-length SS DNA of the parental mutant 173RR/R157A/R158A could be detected after heat denaturation. Unlike the other two rescued mutants, 173RR/R157A/R158A/E46A and 173RR/R157A/R158A/E117A, mutant 173RR/R157A/R158A/E113A displayed no signals at the full-length RC DNA position, while its ssDNA synthesis appeared to be rescued efficiently.

FIG. 5.

In addition to electrostatic interaction, the size (length) of the HBV core protein, in the context of E46A mutation, can also affect viral DNA replication. (A) The same mutation, E46A, was introduced into different core contexts, SVCWT, SVC173GG, SVC171, and SVC169. Although SVC171 has the same charge content as SVC173GG, the former is shorter than the latter by two glycine residues. (B) The mutants were assayed by cotransfection and Southern blot analysis as described in the legend to Fig. 1C. The sizes of RC DNA products appeared to gradually increase when the length of HBc was increased. (Bottom) Mutant capsids collected from cell lysates were as stable as wild-type capsids by Western blot analysis.

FIG. 6.

HBc 1-172 is sufficient for a WT-like DNA phenotype. (A) The truncated core mutant SVC172 contains one arginine at position 172. (B) HBc mutants 171, 172, and 173 and SVC WT were assayed as described in the legend to Fig. 1C. Mutant SVC 172 displayed a WT-like DNA phenotype. (Bottom) Mutant capsids collected from cell lysates were as stable as wild-type HBV capsids by Western blot analysis.

FIG. 7.

Capsid stabilities of wild-type and various HBc mutant capsids isolated from E. coli were compared before and after micrococcal nuclease digestion by native agarose gel electrophoresis. +, with micrococcal nuclease digestion; −, no micrococcal nuclease digestion. (Top) SYBR green II staining for encapsidated RNA. (Bottom) The same gel was destained and restained with Sypro Ruby for HBc capsid protein. The assay procedures are described in detail in reference . The numbers represent the ratios of signals of Sypro Ruby staining for capsid particle-associated proteins with (+) versus without (−) micrococcal nuclease treatment. These numbers reflect in vitro capsid stability in the absence of encapsidated RNA. The results indicated that the capsid stability of 183 WT, 173RR, and 173GG/E117A depends on the presence of encapsidated RNA, while the capsid stability of 173GG, 173GG/E113A, and 173GG/E46A does not depend on the presence of encapsidated RNA.