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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques
  • Cell Line
  • Genetic Complementation Test
  • Hepacivirus / genetics*
  • Hepacivirus / physiology*
  • Host-Pathogen Interactions*
  • Humans
  • Optical Imaging / methods*
  • Proteomics / methods
  • RNA, Viral
  • Replicon*
  • Viral Nonstructural Proteins / genetics*
  • Viral Nonstructural Proteins / physiology
  • Virus Assembly
  • Virus Replication

Substances

  • RNA, Viral
  • Viral Nonstructural Proteins