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

2009 ◽  
Vol 83 (20) ◽  
pp. 10616-10626 ◽  
Author(s):  
Margaret Newman ◽  
Pong Kian Chua ◽  
Fan-Mei Tang ◽  
Pei-Yi Su ◽  
Chiaho Shih
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Electrostatic Interaction ◽  
Micrococcal Nuclease ◽  
Interaction Hypothesis ◽  
Micrococcal Nuclease Digestion ◽  
B Virus ◽  
Nuclease Digestion ◽  
Capsid Stability

ABSTRACT 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.

scholarly journals Underrepresentation of the 3′ Region of the Capsid Pregenomic RNA of Duck Hepatitis B Virus

2004 ◽  
Vol 78 (5) ◽  
pp. 2179-2186 ◽  
Author(s):  
Kristin M. Ostrow ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Complete Recovery ◽  
Biochemical Properties ◽  
Micrococcal Nuclease ◽  
Established Method ◽  
Duck Hepatitis ◽  
Micrococcal Nuclease Digestion ◽  
B Virus ◽  
Nuclease Digestion

ABSTRACT The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes. This report describes differences between the 3′ region and other regions of the pgRNA isolated from capsids. Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3′ region of the pgRNA compared to the 5′ region. This underrepresentation of the 3′ region was partly a result of microccocal nuclease digestion of the 3′ region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3′ region. These results indicate that the 3′ region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3′ region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion. Additional experiments show that the 3′ ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail. These data provide evidence that the 3′ region of the capsid pgRNA has biochemical properties different from those of its 5′ region. Possibly, the 3′ region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion.

Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro

2008 ◽  
Vol 78 (3) ◽  
pp. 242-249 ◽  
Author(s):  
Jun Xu ◽  
Jue Wang ◽  
Fei Deng ◽  
Zhihong Hu ◽  
Hualin Wang
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Green Tea ◽  
Epigallocatechin Gallate ◽  
Green Tea Extract ◽  
B Virus ◽  
Tea Extract

scholarly journals Epitope–Paratope Interaction of a Neutralizing Human Anti-Hepatitis B Virus PreS1 Antibody That Recognizes the Receptor-Binding Motif

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 754
Author(s):  
Jisu Hong ◽  
Youngjin Choi ◽  
Yoonjoo Choi ◽  
Jiwoo Lee ◽  
Hyo Jeong Hong
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Hbv Infection ◽  
Binding Motif ◽  
Alanine Scanning Mutagenesis ◽  
B Virus ◽  
Complementarity Determining Regions

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A–D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19–34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope–paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection.

scholarly journals Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

2004 ◽  
Vol 48 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Radhakrishnan P. Iyer ◽  
Yi Jin ◽  
Arlene Roland ◽  
John D. Morrey ◽  
Samir Mounir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Parallel Synthesis ◽  
Hbv Replication ◽  
Long Term Treatment ◽  
B Virus ◽  
Dna Strand

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

Anti-Hepatitis B Virus Activity in Vitro of Combinations of Tenofovir with Nucleoside/nucleotide Analogues

2009 ◽  
Vol 19 (4) ◽  
pp. 165-176 ◽  
Author(s):  
Yuao Zhu ◽  
Maria Curtis ◽  
Xiaoping Qi ◽  
Michael D Miller ◽  
Katyna Borroto-Esoda
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleotide Analogues ◽  
Virus Activity ◽  
B Virus
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