Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion

Bang, Genie; Kim, Kyun-Hwan; Guarnieri, Michael T.; Zoulim, Fabien; Kawai, Sadaaki; Li, Jisu; Wands, Jack R.; Tong, Shuping

doi:10.1016/j.virol.2004.11.029

Cited by 15 publications

(23 citation statements)

References 40 publications

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“…They have been described previously (1,2). The core Ϫ 4B dimer genome contains a C2044G nonsense mutation in the core gene to ablate core protein expression (1).…”

Section: Mutant Hbv Constructsmentioning

confidence: 99%

“…This construct maintains high replication capacity and efficient HBeAg expression as well as robust expression of HBsAg (hepatitis B surface antigen, viral envelope proteins) and secretion of virus particles. Each mutation was created by overlap extension PCR using Roche High-Fidelity PCR system (1,2,11,20). The PCR products were double digested with either RsrII-BspE1 (positions 1750 to 2328) or RsrII-ApaI (positions 1750 to 2509) for cloning back to the N4 construct.…”

mentioning

confidence: 99%

“…The Pol Ϫ /ε Ϫ 4B genome contains a C2589T nonsense mutation in the polymerase gene to prevent polymerase expression as well as a G1879T/T1880A double mutation in the loop of the ε signal, thus abolishing packaging of pregenomic RNA (2).…”

mentioning

confidence: 99%

“…The Huh7 human hepatoma cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% calf serum. The HBV dimers were transfected into Huh7 cells via the TransIT Transfection system (Mirus) as described previously (2). Serum-free minimal essential medium (150 l) was mixed with 4 l of transit-LT1 reagent, vortexed, and incubated at room temperature for 15 min.…”

mentioning

confidence: 99%

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Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

Guarnieri

Kim

Bang

et al. 2006

J Virol

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The pregenomic RNA directs replication of the hepatitis B virus (HBV) genome by serving both as the messenger for core protein and polymerase and as the genome precursor following its packaging into the core particle. RNA packaging is mediated by a stem-loop structure present at its 5 end designated the signal, which includes the core gene initiator AUG. The precore RNA has a slightly extended 5 end to cover the entire precore region and, consequently, directs the translation of a precore/core protein, which is secreted as e antigen (HBeAg) following removal of precore-derived signal peptide and the carboxyl terminus. A naturally occurring G1862T mutation upstream of the core AUG affects the bulge of the signal and generates a "forbidden" residue at the ؊3 position of the signal peptide cleavage site. Transfection of this and other mutants into human hepatoma cells failed to prove their inhibition of HBeAg secretion but rather revealed great impairment of genome replication. This replication defect was associated with reduced expression of core protein and could be overcome by a G1899A covariation, or by nonsense or frameshift mutation in the precore region. All these mutations antagonized the G1862T mutation on core protein expression. Cotransfection of the G1862T mutant with a replication-deficient HBV genome that provides core protein in trans also restored genome replication. Consistent with our findings in cell culture, HBV genotype A found in African/Asian patients has T1862 and is associated with much lower viremia titers than the European subgroup of genotype A.The hepatitis B virus (HBV) primarily infects the liver and causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. It is an enveloped DNA virus with a small double-stranded genome of 3.2 kb. Inside hepatocytes, viral genomic and subgenomic RNAs are transcribed from a covalently closed circular DNA template in the nucleus and exported to the cytoplasm for translation into viral proteins. The core protein assembles into the core particle, packaging both the pregenomic RNA and DNA polymerase. Subsequently, the DNA polymerase synthesizes the negative-strand DNA via reverse transcription from the RNA template, followed by RNA degradation and synthesis of positive-strand DNA. The core particle with the double-stranded DNA genome is enveloped by host-derived lipids and viral envelope proteins and secreted as an infectious virus particle (for a review, see reference 7). Since the 3.5-kb pregenomic RNA is also the mRNA for the expression of both core protein and DNA polymerase, it is the sole component required for genome replication. Consequently, increased transcription of pregenomic RNA will lead to enhanced HBV replication, as exemplified by the naturally occurring core promoter mutants (3,20).The AUG initiator of the core gene (position 1901 to 1903) is located approximately 80 nucleotides (nt) downstream of the 5Ј end of its mRNA, while the initiation codon of polymerase is 400 nt further downstream. Therefore, core pro...

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“…They have been described previously (1,2). The core Ϫ 4B dimer genome contains a C2044G nonsense mutation in the core gene to ablate core protein expression (1).…”

Section: Mutant Hbv Constructsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

Guarnieri

Kim

Bang

et al. 2006

J Virol

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“…According to the total HBV-DNA load, the patients were divided into two groups: 29 patients with ≤10 5 copies̸ml and 33 patients with >10 5 copies̸ml. The criteria for CHB diagnosis were previously described (11). All the patients were HBsAg-positive and hepatitis C virus-, hepatitis D virus-, human immunodeficiency virus type 1 (HIV-1)-and HIV-2-negative.…”

Section: Subjectsmentioning

confidence: 99%

Expression levels of CD28, CTLA-4, PD-1 and Tim-3 as novel indicators of T-cell immune function in patients with chronic hepatitis B virus infection

et al. 2014

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Abstract. Chronic hepatitis B (CHB) is one of the most common types of infectious diseases worldwide. The interaction between hepatitis B virus (HBV) and the host immune response is vital for the clinical outcome of HBV infection. Costimulatory signals are key factors for the host immune response and play a critical role in innate immunity, particularly antiviral immunity. The aim of the present study was to investigate the correlation between the expression levels of costimulatory molecules and the different states of CHB infection, including the expression levels prior to and following treatment with antiviral agents. The expression levels of CD28, CTLA-4, PD-1, Tim-3 and T-cell subsets were determined by flow cytometry. The load of HBV DNA in the serum was detected by quantitative polymerase chain reaction and the serology markers, including HBeAg and alanine aminotransferase (ALT), were measured by conventional methods. Compared to the healthy control group, the expression levels of CD28 and CTLA-4 on CD4 T cells prior to and following treatment with antiviral agents (the pre-and post-treatment groups, respectively) were significantly decreased, while the expression levels of Tim-3 on CD4 and CD8 T cells were significantly increased. In addition, the expression levels of PD-1 on CD4 and CD8 T cells in the pre-treatment group were significantly increased compared to those in the post-treatment and healthy control groups. Moreover, the multivariate analysis revealed that the levels of ALT and HBV-DNA in the serum were significantly positively correlated with PD-1 expression levels. In conclusion, the expression levels of these costimulatory molecules reflect the immune dysfunction of T cells in patients with CHB and, combined with T-cell subset analysis may be used as a novel evaluation system of immune function in patients with HBV infection. IntroductionChronic hepatitis B (CHB) is one of the most common types of infectious diseases worldwide. It was previously demonstrated that hepatitis B virus (HBV) is not directly cytopathic and that the interactions between HBV and the host immune response are crucial for the clinical outcome of HBV infection (1,2). The main cause of CHB is considered to be a cell immunity function disorder. Briefly, HBV evades the cellular immune response, avoids clearance and may cause persistent viral infections. During this process, T cells play a vital role in the immune function (3). During the process of immune response, the HBV antigens are processed by antigen-presenting cells (APCs) and presented to naïve T cells by major histocompatibility complex (MHC) molecules. Subsequently, these cells deliver a primary signal to initiate T-cell activation by engaging the T-cell receptor̸CD3 complex with foreign antigens associated with MHC molecules (4). The activation of T cells is also regulated by the balance between positive and negative signals. This balance is maintained by the interaction of costimulatory or coinhibitory receptors on T cells and their ligands on APCs. These stimul...

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HBV PreC interacts with SUV39H1 to induce viral replication by blocking the proteasomal degradation of viral polymerase

Hou,

Zhao,

Cai

et al. 2024

Journal of Medical Virology

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Hepatitis B e antigen (HBeAg) seropositivity during the natural history of chronic hepatitis B (CHB) is known to coincide with significant increases in serum and intrahepatic HBV DNA levels. However, the precise underlying mechanism remains unclear. In this study, we found that PreC (HBeAg precursor) genetic ablation leads to reduced viral replication both in vitro and in vivo. Furthermore, PreC impedes the proteasomal degradation of HBV polymerase, promoting viral replication. We discovered that PreC interacts with SUV39H1, a histone methyltransferase, resulting in a reduction in the expression of Cdt2, an adaptor protein of CRL4 E3 ligase targeting HBV polymerase. SUV39H1 induces H3K9 trimethylation of the Cdt2 promoter in a PreC‐induced manner. CRISPR‐mediated knockout of endogenous SUV39H1 or pharmaceutical inhibition of SUV39H1 decreases HBV loads in the mouse liver. Additionally, genetic depletion of Cdt2 in the mouse liver abrogates PreC‐related HBV replication. Interestingly, a negative correlation of intrahepatic Cdt2 with serum HBeAg and HBV DNA load was observed in CHB patient samples. Our study thus sheds light on the mechanistic role of PreC in inducing HBV replication and identifies potential therapeutic targets for HBV treatment.

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Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion

Cited by 15 publications

References 40 publications

Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

Point Mutations Upstream of Hepatitis B Virus Core Gene Affect DNA Replication at the Step of Core Protein Expression

Expression levels of CD28, CTLA-4, PD-1 and Tim-3 as novel indicators of T-cell immune function in patients with chronic hepatitis B virus infection

HBV PreC interacts with SUV39H1 to induce viral replication by blocking the proteasomal degradation of viral polymerase

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