Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nucleaseresistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.The hepatitis B virus (HBV) core antigen (HBcAg) is a 22-kDa protein with multiple functions, including interactions with the pregenomic RNA (pgRNA) and the polymerase during encapsidation (10,31,41) and with the viral DNA during reverse transcription and DNA elongation (18), polymerization to form the nucleocapsid or core particles (4, 13), importing of HBV DNA to the nucleus (51), and targeting to the endoplasmic reticulum for envelope formation (5). The C-terminal region of HBcAg contains a protamine-like domain, which is rich in arginine and presumably binds to HBV RNA and DNA during pgRNA encapsidation and DNA replication (13,18,35). This arginine-rich domain has been shown to be dispensable for core particle assembly but not for viral replication (2, 4, 13, 30).
To test a previously coined "charge balance hypothesis" of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecularweight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined "electrostatic interaction hypothesis" is discussed.Chronic infection with hepatitis B virus (HBV) leads to the development of cirrhosis and hepatocellular carcinoma (6,31,36). HBV core protein (HBc) consists of the assembly domain (HBc amino acids 1 to 149) at the N terminus and the argininerich domain (ARD) at the C terminus (HBc amino acids 150 to 183) (33,34). Escherichia coli-expressed HBc can spontaneously self-assemble into 28-nm capsid particles with a spherical appearance indistinguishable from that of human liver-derived capsid particles (7). Such capsid particles have been shown to package RNAs transcribed in E. coli (5,8,11,28,37). The four-helix bundle structure of HBV capsid particles is based on cryo-electron microscopy and X-ray crystallography using Cterminally truncated HBc (34, 38). At present, there is no known structure at the C terminus of HBc capsids (34, 41). HBc amino acids 150 to 183 contains four stretches of clustering arginine residues (ARD-I, -II, -III, and -IV) (Fig. 1). When the C-terminal domain of hepadnaviral core protein was serially truncated, a viral replication defect was observed (4,19,22,27,39). To date, it remains to be elucidated why the C term...
Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75°C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T3؍ or T4؍ icosahedral particles. No difference in the ratio between T3؍ (78%) and T4؍ particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T3؍ and T4؍ particles successfully separated by using a novel agarose gel electrophoresis procedure.
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 postulat...
Decidual and placental relaxins have been proposed as autocrine/ paracrine hormones in the remodeling of collagen in the amnion and chorion in the last weeks of pregnancy. The matrix metalloproteinase-1 (MMP-1) is a key enzyme in the degradation of the interstitial collagens which predominate in the fetal membranes. Distribution of the MMP-1 gene and of the MMP-1 protein was shown by in situ hybridization and immunolocalization, respectively, in amnion, chorion, and decidua collected from patients before the onset of spontaneous labor. The distribution of MMP-1 in the chorionic cytotrophoblast and decidua coincided with that of the human relaxin receptor, detected by tissue section autoradiography in tissues collected at the same stage of pregnancy. Fetal membrane explants were used to study the effect of exogenous human relaxin H2. These responded by a dose-dependent increase in expression of the MMP-1 gene, in its secreted protein, and in its enzyme activity in the medium. A similar dose-dependent increase in the tissue plasminogen activator (tPA) gene and protein upon exposure of the explants to relaxin H2 suggested a coordinated cascade system, resulting in increases in secreted activities of MMP-1, MMP-3 (stromelysin), and MMP-9 (gelatinase B). There was no effect on the genes or proteins for MMP-2 (gelatinase A) or tissue inhibitor of metalloproteinase-1 (TIMP-1), showing the specificity of the response. This coordinated regulation by relaxin H2 of tPA, MMP-1, MMP-3, and MMP-9 would result in more complete degradation of the fetal membrane extracellular matrix components.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.