The hepatitis В virus: general description, physical structure, genetic organization, gene transcripts and genomic regulatory elements

The HBV genome is a circular partially double-stranded DNA molecule. It contains four overlapping open reading frames (ORFs) genes. The S (preS1, preS2) region(s) encodes the major (small), middle and large proteins (HBsAg). The C and pre-C regions encode HBcAg and HBeAg. The X region encodes a polypeptide expressed during HBV infection. The P region codes for a protein with several, functions in replication. Four classes of HBV mRNAs have been identified, in the HBV genome the pre-S1 promoter expresses the large protein. The pre-S2/S promoters produce both major and middle proteins. The X promoter produces 0.7 and 0.9 kb transcripts. The C and pre-C promoters produce the Core, pol(HBcAg), and the HBeAg proteins. Enhancer I and II are key regulatory elements in the transcriptional regulation of HBV. The activity of enhancer II is highly the liwr specific. Enhancer II activates the transcriptional activity of both the pre-S1 and preS2/S promoters. Two HBV enhancers strongly affect the activity of all three major HBV promoters. A box-α in the II-A and box-β in II-B elements of HBV genome, are necessary for the enhancer II function. Either box-α or box-β can regulate the activity of the Core promoter, a, b, f proteins and c, d proteins bind to box-α and box-β, respectively, and mediate the enhancer function.Геном вірусу гепатиту В людини (HBV) існує у вигляді дволанцюгових кільцевих молекул ДНК Він містить чотири фланкуючі відкриті рамки зчитування (ORFs) генів, S (preSl, preS2) регіоні и) кодує головний, середній та великий білки (HBsAg). С і preC регіони кодують HBcAg і HBeAg, X регіон кед у є поліпептид, який експресується за час HBV інфекції Р регіон кодує білок з різноманітними функціями у реплікації. Ідентифіковано чотири класи HBV мРНК. У геномі HBV pre-S1 промотор продукує великий білок, pre-S2/S виробляє головний і середній білки. X промотор кодує 0,7 і 0,9 трагскрипти. С і пре-С промотори кодують Core і pol(HBVcAg) і HBeAg білки. Ключовими регуляторними елементами HBV є енхансери І і II. Активність енхансера II є специфічною для печінки. Він активує транскрипцію обох пре-S1 і пре-S2/S промоторів. Два HBV енхансери активують основні промото­ри HBV. Бокс-α в І 1-А і бокс-β в ІІ-В елементах у геномі HBV необхідні для функції енхансера II. Бокси а і р можуть регулювати активацію промотора Core, білки a, b, f і білки с, d прикріплюються у боксах а і р відповідно і впливають на енхансерну функцію.Геном вируса гепатита В человека (HBV) существует в виде двухцепочных кольцевых молекуул ДНК Он содержит четыре фланкирующие открытые рамки считывания (ORF8) генов. S (преS1, преS2) регион/ы) кодирует главный, средний и боль­шой белки (HBsAg). С и пре-С регионы кодируют HBсAg и HBeAg. X регион кодирует полипептид, экспрессирующийся в течение HBV инфекции. Р регион кодирует белок с разнообраз­ными функциями в репликации. Идентифицированы четыре класса HBV мРНК. В геноме HBV npe-Sl промотор продуци­рует большой белок, пpe-S2/S промотор производит оба (главный и средний) белка. X промотор кодирует 0,7 и 0,9 транскрипты. С и пре-С промоторы кодируют Core и pol(HBcAg) и HBeAg белки. Ключевыми регуляторными эле­ментами в HBV являются энхансеры I и II. Активность энхансера II очень специфична для печени, Энхансер II активи­рует транскрипционную активность пре-S1 и пре-S2/S про­ моторов. Два HBV энхансера активируют основные промото­ры HBV. Вокс-α в НА и бокс-β в П-В элементах в геноме HBV необходимы для функции энхансера II. Воксы α и β могут регулировать активацию Core промотора, белки a, b, f и белки с, d прикрепляются в боксах α и β соответственно и дейст­вуют на энхансерную функцию

The gene S promoter of hepatitis B virus confers constitutive gene expression.

The properties of the promoter of the hepatitis B surface antigen (HBsAg) were studied using recombinants containing either this promoter or the SV40 early promoter. Mouse L cells were transfected with these recombinants and the levels of gene expression obtained with the two promoters were compared. The level of expression of a cellular gene, the human fibroblast interferon gene, obtained with the HBsAg promoter was comparable to that obtained with the SV40 early promoter. Similarly when the HBsAg gene was controlled by the SV40 early promoter the level of HBsAg synthesis is in the same range as that observed with its own promoter. Together these results suggest that although the HBsAg gene codes for a structural viral protein, its expression is constitutive as for an early gene. The implications of these observations on the synthesis of HBV particles in vivo are discussed

The effect of a valine to phenylalanine mutation in the precore region of hepatitis b virus on virus replication, HBeag maturation and expression

Hepatitis B virus (HBV) infection is endemic in South Africa. A unique feature of HBV carriers in this geographical region is that majority of the carriers are HBV e antigen (HBeAg) negative before they reach adulthood. Up to a few years ago the reason for this early loss of HBeAg was unknown. HBeAg is translated from the precore mRNA whose transcription is controlled by the basic core promoter. The dominant subgenotype of HBV in South Africa is subgenotype A1. This subgenotype is characterized by various variations/mutations in the basic core promoter and precore region of HBV that can affect HBeAg expression. Within the basic core promoter, A1762T/G1764A mutations can affect the expression of HBeAg at the transcriptional level. These mutations interfere with transcription factor binding to the basic core promoter and suppress the transcription of precore mRNA that is translated into HBeAg, hence reducing HBeAg expression. Mutations at nucleotides 1809-1812, also within the basic core promoter, reduce HBeAg expression at the translational level by creating a “sub-optimal” Kozak sequence upstream from the precore start codon at position 1814 from the EcoRI site. Following translation of the precore/core fusion protein, this precursor molecule of HBeAg is post-translationally modified by signal peptide cleavage at a fixed site on the amino end and at variable sites on the carboxyl end. The precore/core open reading frame on the precore mRNA that codes for the precursor of HBeAg, overlaps the region that codes for the encapsidation signal (ε) on the pregenomic RNA (pgRNA). pgRNA plays a pivotal role in the initiation of reverse transcription and is translated into the capsid protein and the polymerase enzyme. In previous studies, a guanine (G) to thymine (T) mutation at nucleotide 1862 within the precore region was identified in subgenotype A1 isolates from asymptomatic carriers of the virus and from hepatocellular carcinoma patients from South Africa. This mutation could conceivably have two functional consequences. Firstly, the G1862T mutation could change the secondary structure of ε and could interfere with and hence affect HBV replication. Secondly, the phenotypic change from valine to phenylalanine introduced by the G1862T mutation at codon 17 (-3 position to the signal peptidase recognition motif) is close to the signal peptide cleavage site at position 19 (-1 position to the signal peptidase recognition motif), and may therefore abrogate signal peptide cleavage. Therefore the objective of this study was to functionally characterize the HBV G1862T mutation and its equivalent G1982T found in woodchuck hepatitis virus (WHV). This was done by determining the effect of this mutation on viral replication and eAg expression of plasmid constructs in vitro. Replication competent clones were constructed by mutating the wild-type of HBV and the mutant of WHV. The G1862T and T1982G mutation were introduced into the precore region of replication competent HBV and WHV plasmids, respectively, by site-directed mutagenesis. HBeAg-expression and WHeAg-expression plasmids were constructed using the replication competent clones as templates. For HBV, the templates used belonged to genotype D or to genotype D in which the precore region was mutated into a genotype A context, genotype ‘A’. Huh 7 hepatoma cells were transfected with the respective replication competent clones and HBV replication was followed using Southern hybridization and real time polymerase chain reaction (PCR). The secretion and expression of HBeAg were monitored using enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and confocal microscopy, following transfection with the eAg expressing plasmids. The secretion and expression of WHeAg were monitored using pulsed radioactivelabel, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunocytochemistry and confocal microscopy. HBV replication was significantly reduced when the G1862T was introduced into genotype D but not into genotype ‘A’ HBV replication competent constructs. Following transfection with mutated HBeAg-expression plasmids, a reduction of 38 % for genotype D, and 54 % for genotype ‘A’ in HBeAg secretion relative to the wild-type were observed. Using the WHV constructs, reduced processing of the mutant relative to the wild-type protein was demonstrated using pulse-radioactive labelling. Using confocal microscopy it was demonstrated that both the mutant HBeAg and mutant WHeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum Golgi intermediate compartment and Golgi. This accumulation is because the introduction of a phenylalanine at position -3 of the signal peptide cleavage site interfered with the post-translational modification of the HBeAg precursor protein. The aggregates of mutant HBV protein increased in size following treatment of cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes, and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. Aggresomes formed by the HBV mutant precore protein resembled Mallory-Denk bodies which are histological and potential markers of progressive liver diseases