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Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection

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Abstract

Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1–E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag–Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.

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Acknowledgments

The authors acknowledge and express our sincere appreciation to Dr. Ishtiaq Qadri for providing the phCMV-5349 and pTG-Luc126 vectors. This study was partially supported by Higher Education Commission (HEC) of Pakistan.

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There are no conflict of interest.

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Authors and Affiliations

  1. Centre of Applied Molecular Biology, Ministry of Science & Technology Govt. of Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore, Pakistan

    Shazia Rafique & Muhammad Iqbal

  2. Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Thokar Niaz Baig, Lahore, 53700, Pakistan

    Muhammad Idrees

  3. Department of Biotechnology, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan

    Amjad Ali

  4. Department of Biotechnology and Biochemistry, University of Veterinary Sciences and Animal Sciences, Lahore, Punjab, Pakistan

    Kashif Iqbal Sahibzada

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  1. Shazia Rafique
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  2. Muhammad Idrees
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Correspondence to Muhammad Idrees.

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Rafique, S., Idrees, M., Ali, A. et al. Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection. Mol Biol Rep 41, 3813–3819 (2014). https://doi.org/10.1007/s11033-014-3247-x

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