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J Virol Methods. 2017 Aug;246:42-50. doi: 10.1016/j.jviromet.2017.04.009. Epub 2017 Apr 21.

Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics.

Author information

1
Institute of Biochemistry of the Romanian Academy, Bucharest, Romania.
2
Institute of Biochemistry of the Romanian Academy, Bucharest, Romania; Department of Anatomy, Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania.
3
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France.
4
Institute of Biochemistry of the Romanian Academy, Bucharest, Romania. Electronic address: pop@biochim.ro.

Abstract

Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety.

KEYWORDS:

Hepatitis C Virus; Live cell imaging; Protein–protein interaction; Proteomics; Virus-host interaction; trans-Complementation

PMID:
28438609
DOI:
10.1016/j.jviromet.2017.04.009
[Indexed for MEDLINE]

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