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Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay
Richard A Urbanowicz1,2, C Patrick McClure1,2, Barnabas King1,2
1School of Life Sciences, The University of Nottingham, Nottingham University Hospitals NHS Trust, Nottingham, UK.
The Journal of General Virology
|July 8, 2016
Summary
Optimizing pseudovirus production is crucial for accurate viral entry studies. Empirical adjustments to plasmid ratios and constructs are essential for reliable phenotyping of hepatitis C virus (HCV) and ebolavirus pseudoviruses (EBOVpv).
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Retrovirus pseudotypes are valuable models for studying enveloped virus entry.
- Hepatitis C virus pseudoparticles (HCVpp) are used for studying HCV entry and screening antivirals.
Purpose of the Study:
- To investigate factors limiting phenotyping of patient-derived HCV glycoproteins using pseudoviruses.
- To establish optimal production protocols for HCVpp and ebolavirus pseudoviruses (EBOVpv).
Main Methods:
- Empirical optimization of plasmid ratios (packaging construct and glycoprotein-encoding plasmid).
- Assessing the impact of different retroviral packaging constructs on pseudovirus function.
- Validating findings using chimeric cell-cultured HCV and ebolavirus pseudoviruses.
Main Results:
- Genetically related HCV glycoproteins exhibit varied behavior in pseudovirus entry models.
- Successful HCVpp genesis requires empirical optimization of plasmid ratios for different clones.
- Pseudovirus production is sensitive to plasmid amounts, ratios, and specific glycoprotein constructs.
- Previously non-functional glycoproteins were found to mediate entry upon optimization.
Conclusions:
- Empirical optimization of pseudovirus production is critical for each specific glycoprotein sequence.
- This optimization is essential for achieving optimal titers and accurate phenotyping in viral entry studies.
- The findings apply to both HCV pseudoparticles and ebolavirus pseudoviruses.
