Heteroaryldihydropyrimidines（HAPS）抑制HBV核衣壳的成熟与cccDNA转录
CONTROL ID: 1426205
PRESENTATION TYPE: Oral or Poster
CURRENT CATEGORY: Hepatitis B
CURRENT DESCRIPTORS: I02. Treatment and Clinical Trials
TITLE: Heteroaryldihydropyrimidines (HAPs) inhibit HBV replication in tissue culture by targeting both nucleocapsid maturation and cccDNA transcription
AUTHORS (FIRST NAME, LAST NAME): Laura Belloni1, 3, Lichun Li4, Gianna Aurora Palumbo1, 2, Srinivas Reddy Chirapu 5, Ludovica Calvo1, 3, M. G. Finn5, Adam Zlotnick4, Massimo Levrero1, 2
Institutional Author(s):
INSTITUTIONS (ALL): 1. Dept of Internal Medicine, Sapienza University, Rome, Italy.
2. Life Nanosciences Laboratory, Sapienza University, Rome, Italy.
3. EAL Inserm U785, Sapienza University, Rome, Italy.
4. Department of Molecular & Cellular Biochemistry , Indiana University, Bloomington, IN, United States.
5. Dept Chemistry, Scripps Research Inst, La Jolla, CA, United States.
ABSTRACT BODY: Background: The development of novel combination based therapies for HBV infection requires new antivirals that block viral life cycle functions other than those associated with the viral polymerase. The HBV Core, that comprises the viral capsid, nucleic acid, and host and viral ancillary proteins, represents an attractive target. Proper assembly of the capsid is critical for RNA packaging, reverse transcription, and intracellular trafficking. Moreover, core proteins (Cp) have been shown to interact with histones and to bind the nuclear cccDNA, possibly contributing to the regulation of cccDNA function and the maintainance of the cccDNA stability. Hetero-aryl-dihydropyrimidines (HAPs) are a new class of antivirals inhibiting HBV replication in vitro and in vivo. HAPs enhance the rate and the extent of core protein (Cp) assembly over a broad concentration range and act as allosteric effectors to induce an assembly-active state or, at high concentration, stabilize preferentially non-capsid polymers of Cp. Here we investigated the impact of HAP12 on cccDNA formation, levels and transcription as part of its antiviral activity against HBV.
Methods: Capsid-associated HBV-DNA (TaqMan real-time PCR), cccDNA (TaqMan real-time PCR) and pgRNA levels (quantitative real-time PCR with specific primers), were assessed both in HepG2 cells transfected with full length HBV genomes and in a HepG2 stable clone that accumulates cccDNA, left untreated or treated with the hetero-aryl-dihydropyrimidine HAP12 at 1-5 microM.
Results: We confirmed, using the cccDNA ChIP assay, that Cp is recruited onto the cccDNA in HBV replicating cells. HAP12 treatment of cells transfected with wild type linear HBV genomes showed a complete suppression of HBV replication at 72 and 96 hrs with a peak >50% reduction of pgRNA transcription at 96 hours, without significant changes in cccDNA levels. In the HepG2 stable cell line we confirmed the strong HAP12 inhibitory effect on pgRNA transcription and HBV replication as well as the lack of significant effect on steady state cccDNA levels. By treating the cells with HAP12 already before the establishment of the cccDNA pool we could also show that cccDNA formation and accumulation are not targeted by HAP12.
Conclusions: HAPs binding to HBV capsid, in addition to conformational change resulting in a core protein that does not support HBV replication, also target the nuclear cccDNA. We show that inappropriate assembly and effective cytoplasmic trapping of Cp induced by HAPs affects, through mechanisms that have not been yet clarified, cccDNA function rather than the formation of cccDNA or the establishment/maintenance of the cccDNA nuclear pool. 作者: 肝胆速递 时间: 2012-10-8 17:52
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