Testing the Balanced Electrostatic Interaction Hypothesis of Hepatitis B Virus DNA Synthesis by Using an In Vivo Charge Rebalance Approach

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.Human hepatitis B virus (HBV) is an important human pathogen (5, 27, 34) that can replicate via an RNA intermediate (31, 33). Wild-type (WT) HBV core protein (HBc) is 183 amino acids long (adr and ayw subtypes) and consists of two distinct domains connected by a hinge region. The assembly domain spans amino acids 1 to 140, and the arginine-rich domain (ARD) spans amino acids 150 to 183. The ARD of HBc 150-183 is not required for capsid assembly in Escherichia coli (4, 8, 10, 21, 35). During nucleocapsid (capsid) formation, the HBc protein assembles into an icosahedral particle via a dimer intermediate (32). The ARD of HBc is known to be capable of binding to nucleic acids (12, 25). Serine phosphorylation at the C terminus of HBc is known to be important for RNA encapsidation, DNA synthesis, and virion secretion (2, 11, 14, 15, 17, 19, 24, 26, 39, 40). To date, there is no structural information available for the C terminus of HBc in capsids (32). The 4-helix bundle structure of HBc capsids is based on a C-terminally truncated capsid protein, HBc149 (6, 7, 36). Our research progress in the study of HBV biology has been hampered due to the lack of structural information about the HBc C-terminal tail, which plays an important regulatory role throughout the life cycle of HBV.Recently, we proposed a hypothesis that “charge balance” could be important for HBV capsid stability, assembly, RNA encapsidation, and DNA replication (17). This hypothesis postulates that many important viral activities could be influenced by the electrostatic interaction between the positive charge of the basic amino acid-rich domains of a nucleocapsid protein and the negative charge of the nucleocapsid-associated nucleic acids (17). Previously, we and others demonstrated that a mutant HBc 164, which lacks a total of eight arginine residues at the C terminus, can package both the nonspliced 3.5-kb pregenomic RNA (pgRNA) and the 2.2-kb spliced RNA (14, 17). However, pgRNA encapsidated by HBc 164 is RNase sensitive, while the encapsidated 2.2-kb spliced RNA is RNase resistant. The pgRNA and spliced RNA encapsidated by the full-length wild-type HBc 183 is also RNase resistant (14, 17). This result provided us with the first clue to invoke the previously coined “charge balance hypothesis” of RNA encapsidation and capsid stability (17). In subsequent studies, we added arginines back to the truncated HBc 164 gradually and noted that core particle-associated viral DNA also increased gradually in both size and intensity. This result provided us with the second clue to expand the previous hypothesis from RNA encapsidation to include viral DNA synthesis (17). Instead of totally nonquantitative or nonspecific binding between nucleic acids and a basic protein, our electrostatic-interaction hypothesis entails a stoichiometry-like concept between basic residues and their binding partner of nucleic acids. One of the reasons for such a demand for a more quantitative charge-charge interaction could be related to the putative intersubunit positive-charge repulsion built into the context of an icosahedral particle (22).We and others demonstrated previously that truncated HBc 173 (also denoted HBc 173RR in Fig. ​Fig.1)1) is sufficient for a WT-like DNA phenotype (17, 20). Mutant 173GG, containing two substitutions from arginine (R) to glycine (G) at codons 172 and 173 of HBc 173, exhibited a shorter-than-full-length HBV DNA phenotype, suggesting the importance of sufficient positive charge for viral DNA synthesis (17).Open in a separate windowFIG. 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.To test further the effect of electrostatic interaction on viral DNA synthesis, we asked if one could restore the DNA replication defect of the arginine-deficient ARD IV mutant 173GG or ARD II mutant 173RR/R157A/R158A by reducing their negative charges at specific positions. Indeed, our results showed that a single substitution from glutamic acid to alanine (E-to-A) at position 46 or 117, and to a lesser extent at position 113, could restore the WT-like DNA replication in the context of ARD IV mutant 173GG. Similarly, in the context of ARD II mutant 173RR/R157A/R158A, both E46A and E117A could rescue the full-length single-stranded DNA (ssDNA) and relaxed-circle (RC) DNA syntheses.Using an in vitro capsid assembly/disassembly assay, we demonstrated that the capsid stability of both wild-type full-length HBc 183 and truncated HBc 173 capsid particles from E. coli are dependent on the presence of encapsidated RNA (22). Upon treatment with micrococcal nuclease, encapsidated RNAs were digested and lost, leading to capsid disassembly. In this study, we demonstrated that mutant 173GG maintained capsid stability upon the loss of encapsidated RNA, probably due to the loss of intersubunit positive charge repulsion at ARD IV. Interestingly, when mutation E117A was introduced into the context of mutant 173GG, the capsid stability of mutant capsids E117A/173GG was once again dependent on the encapsidated RNA in a manner similar to that of its parental mutant, 173. Further studies also revealed that the truncated HBc mutant 172, like mutant 173, is sufficient for HBV DNA replication. Taken together, these results lend strong support to the balanced electrostatic-interaction hypothesis of HBV DNA replication.