Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo A.; Yi, Min Kyung; Lemon, Stanley M.; Benhar, Itai; Tur–Kaspa, Ran

doi:10.1016/j.antiviral.2010.08.001

Cited by 17 publications

(13 citation statements)

References 47 publications

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“…The Clonetics Hepatocyte Cell Culture System containing normal primary human hepatocytes (PHH) (Lonza) were cultured as described previously [52]. The HCV-N NS3-NS5B subgenomic replicon cell line included in this study was cultured as we previously described [53].…”

Section: Methodsmentioning

confidence: 99%

“…Virus stocks were produced in Huh7/FT3-7 cells and viral titers were determined by FFU assay in Huh-7.5 cells, as described previously (39). For all experiments with Huh7.5 or Huh7.5-HS, cells were infected with HJ3-5 chimeric virus at a MOI of 0.1 or 0.5 and passaged for at least 2 weeks until approximately 100% of the cells were HCV positive, determined by immunofluorescence, as previously described [53]. PHH (Lonza) were infected with cell culture infectious HCV HJ3-5 at high MOI (0.5–1) to ensure high efficiency of infection, as described previously [52].…”

Section: Methodsmentioning

confidence: 99%

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Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals

et al. 2019

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The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic “scarring” of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals

et al. 2019

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“…The inhibitory effect observed upon expression of an NS3-specific intrabody was maintained even in the presence of point mutations that confer resistance to small-molecule drugs. As hypothesized by the authors, resistance to antibody-based drugs might occur more slowly than resistance to small-molecule-based drugs because antibodies contact their targets over a comparably large surface area via multiple residues [ 270 ].…”

Section: Applications In Viral Infectionsmentioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

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“…At present, most research has mainly used antibody coding genes in commonly expressed eukaryotic carrier. However, these carriers do not possess the ability to infect cells, and they need an supporting method for inoculation into cells, such as Lipofectamine carrying an antibody-coding plasmid to infect cells, or the use a particular injection device to guide antibodies into cells [3,4].…”

Section: Backgroudmentioning

confidence: 99%

Preparation and bioactivity of anti-Newcastle disease virus-phosphoprotein cytoplasmic transduction peptide antibody

Wang²,

Zhu³

2020

Preprint

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On the basis of cell-penetrating peptide’s character that it can penetrate cytomembrane and transfer macromolecular protein to cytoplasm so to play biological function, we took the experiments. The fuse penetrating peptide our experiment adoptted is HIV-TAT derived fragment-CTP512, with good transmember effect and distinct cytoplasm-position. In this chapter, the research of transmembrane character was processed first. According to the tests on trans- member protein with different concentrations, the best trans-member concentration is 3µM. Afterwards, we found that the location of trans-member antibody is overlapping with phosphoprotein using indirect immunofluorescence test analysis. According to MTT test, there is no significant difference between CTP fusion protein and control on cell proliferation and viability. TCID50 test was used to detect the protective effect of trans-member antibody on cell. Result showed that trans- member antibody has significant cell protection effect compared to the control in the order: ZL.103>ZL.17>Control. Fluorogenic quantitative PCR result showed that trans- member antibody can disturb the duplication and transcription of Newcastle disease virus. This results not only paved a good way to research the transport of disease related protein, but also provide a splendid tool on protein function research.

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Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Cited by 17 publications

References 47 publications

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals

Hepatitis C virus leaves an epigenetic signature post cure of infection by direct-acting antivirals

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

Preparation and bioactivity of anti-Newcastle disease virus-phosphoprotein cytoplasmic transduction peptide antibody

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