Conference 2021 Pre-Recorded Video
Flow Virometry as a Method to Phenotype Viral Surface Proteins
Authors and Affiliations
Jonathan Burnie1,2, Vera A. Tang3, Joshua A. Welsh 4, Arvin T. Persaud1, Laxshaginee Thaya1,2, Jennifer C. Jones4, Christina Guzzo1,2
1. Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada
2. Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada
3. Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Flow Cytometry and Virometry Core Facility, Ottawa, ON K1H 8M5, Canada
4. Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
The human immunodeficiency virus (HIV) contains only one viral surface protein, the envelope glycoprotein (gp120). As the sole viral protein on the viral surface, the majority of HIV vaccine designs have centered on gp120 to induce immune responses. However, HIV contains a plethora of cellular/host proteins on its surface which become incorporated in the HIV envelope as viruses bud from the plasma membrane of infected cells. While most of these proteins remain largely understudied, select cellular envelope proteins are known to play important roles in HIV infection, and may serve as new antiviral targets in vaccine and therapeutic drug designs. Despite the importance of characterizing surface proteins on HIV, current techniques available for this purpose are largely semi-quantitative and do not support high-throughput analysis. Herein, we describe the use of flow cytometry – a technique traditionally used for rapid, high-sensitivity characterization of single cells – to detect and characterize surface antigens on individual HIV particles. Flow cytometry is termed flow virometry here for its specific application to viruses.
HIV particles containing high levels of the cellular proteins integrin α4β7, CD14 or CD162 in the viral envelope were produced in HEK293 cells by transfection. Virus-containing cell culture supernatants were stained for the aforementioned cellular antigens and CD81 using fluor-labeled monoclonal antibodies, and then acquired on a flow cytometer. Using reference beads that were run alongside the viruses, light scatter and fluorescence calibrations were performed after sample acquisition using FCMPass software to present data in standardized units. Data calibration enables comparisons of our quantitative data to those acquired on different instruments, which will aid standardization efforts in this field.
For the first time, we report quantitative estimates of the number of three cellular proteins on individual HIV particles in standardized units (molecules of equivalent soluble fluorophore), with a range of 20-100 cellular proteins per virion. Dual labeling of α4β7, CD14 or CD162 and CD81 demonstrated the capacity for detection of multiple antigens with this system.
Our results suggest that flow virometry can be used to provide high-throughput quantitative, characterization of proteins in the HIV envelope which may prove useful in identifying new viral targets for therapeutic strategies.