Testing an Electrostatic Interaction Hypothesis of Hepatitis B Virus Capsid Stability by Using an In Vitro Capsid Disassembly/Reassembly System

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-molecular-weight 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 arginine-rich 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 C-terminally 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. ​(Fig.1).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 terminus is so important for diverse biological activities, including RNA encapsidation and DNA replication. To address this issue, we proposed previously a so-called “charge balance hypothesis” (22), which highlights the importance of adequate electrostatic interactions between positive charge (basic residues) from HBc and negative charge from encapsidated RNA or DNA.Open in a separate windowFIG. 1.Effects of micrococcal nuclease treatment on HBV nucleocapsids with serially truncated C termini of core proteins. A series of HBV core expression vectors with different lengths and arginine contents were constructed in pET-Blue-1. The truncations are illustrated in the top panel, and the four ARDs (ARD-I, -II, -III, and -IV) are underlined. All constructs self-assembled into capsids when expressed in E. coli. The capsids run as a distinct band on 1% native agarose gels and can be stained with EtBr (upper panel) and Coomassie blue (middle panel). Untreated controls without micrococcal nuclease were incubated with the same buffer and conditions as for micrococcal nuclease-digested samples. In the upper panel, note the loss of the EtBr signal when the encapsidated RNAs were digested by micrococcal nuclease. In the middle panel, we observed three different groups of mutants with three different CBS patterns. Group 1 mutants, including HBc mutants 149, 154, and 157, exhibited no significant change in CBS banding pattern before or after micrococcal nuclease digestion. In group 2 capsids 159, 164, 169, and 171 (#), the CBS banding pattern became more diffuse, less intense, and slightly (yet reproducibly) upshifted. * indicates the near-complete loss of CBS banding in group 3 capsids 167, 173, 175, and 183. To confirm that this loss of CBS probably results from a loss of structural integrity of the capsid particle, rather than from a loss of the core protein per se, aliquots of samples were also run on denaturing SDS-PAGE (bottom panel).The first clue that such an electrostatic interaction could play an important role in RNA encapsidation is from the study of an engineered mutant, HBc 164. Despite its reduced arginine content relative to the wild-type (WT) full-length HBc 183 (Fig. ​(Fig.1),1), HBc mutant 164 can encapsidate, as efficiently as WT HBV, both 3.5-kb pregenomic RNA (pgRNA) and a spliced 2.2-kb subgenomic RNA (sgRNA) (19, 22). When WT HBV nucleocapsids (capsids) were treated with micrococcal nuclease, the encapsidated 3.5-kb pgRNA and its reverse-transcribing RNA template were resistant to micrococcal nuclease treatment. In contrast, when mutant 164 capsids were treated with micrococcal nuclease, the encapsidated 3.5-kb RNA was highly nuclease sensitive, while the encapsidated 2.2-kb sgRNA appeared to be nuclease resistant (19, 22).To further elucidate the mechanism behind this phenomenon, we hypothesized that this result could be due to an abnormal or less stable capsid structure generated by a charge imbalance (insufficient positive charge or excessive negative charge) when arginine-deficient mutant 164 encapsidates the full-length 3.5-kb pgRNA. In contrast, a more normal or stable capsid structure can be generated when arginine-deficient mutant 164 encapsidates the 2.2-kb sgRNA (with a reduced negative charge content relative to the 3.5-kb pgRNA) (22). In addition to RNA encapsidation and capsid stability, electrostatic interaction could also play a role in HBV DNA synthesis and genome maturation. For example, when the truncated C terminus of HBc 164 was progressively restored, the core-associated viral DNA gradually increased in both size and signal intensity (22).In this study, we designed an in vitro capsid disassembly/reassembly experiment which is complementary to the in vivo approach in tissue culture (22; P. K. Chua, F. M. Tang, J. Y. Huang, C. S. Suen, and C. Shih, submitted for publication). We investigated the relationship between the arginine content of HBV capsids and the encapsidated nucleic acid in maintaining capsid stability in vitro. In addition, we demonstrated that HBV capsids that are disassembled by depletion of encapsidated RNA can be efficiently reassembled in the presence of exogenous nucleic acid and non-nucleic acid polyanions but not in the presence of polycations or low-molecular-weight (low-MW) anions. Capsid disassembly induced by RNA digestion appears to be related to intersubunit charge repulsion between ARD-III or ARD-IV in close contact.     

A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the V_max and k_cat values.     

Editorial

Editorial

Editorial     

Roles of db-cAMP,IBMX and RA in Aspects of Neural Differentiation of Cord Blood Derived Mesenchymal-Like Stem Cells

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson''s disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage, there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here, we show that MSCs derived from human umbilical cord blood (MSC^hUCBs) are capable of expressing tyrosine hydroxylase (TH) and Nurr1, markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSC^hUCB. Furthermore, functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms.     

Enterovirus71 (EV71) Utilise Host microRNAs to Mediate Host Immune System Enhancing Survival during Infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Immune complexes and human neoplasia

Summary We have investigated the membrane-binding properties of fetal liver cells (FLC) and developed an assay to quantitate circulating immune complexes (CIC) based on complement (C) receptor binding on FLC. Both binding and blocking studies identified FLC membrane receptors for IgG-Fc, C, and antifetal antibodies (FL-Ab), but not IgG F(ab)2. Fc binding of IgG or aggregated human IgG (AHG) was relatively weak, with an association constant of 1.5×10⁷ l/mol. In contrast, there was a six- to seven-fold increase in binding of AHG by C receptors, with an association constant of 10⁸ l/mol. A simple and sensitive procedure for detecting CIC in the sera of patients with various disease states has been developed by the use of C receptors on FLC. Reference to an AHG standard curve permits quantitation of CIC in micrograms of AHG equivalent per milliliter of serum. Clinical evaluation in patients with active collagen vascular disease and in cancer patients confirmed the reliability, specificity of binding, and sensitivity of the FLC method. Although there was overall agreement with the Raji cell method for CIC detection, FLC-RIA quantiation of CIC was found to be more sensitive than the Raji cell assay. Other discrepancies could be explained by differing sensitivity to CIC size.Preliminary results were presented at the Seventieth Annual Meeting of the American Association for Cancer Research, May 1979 [40]