Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

Abstract

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

On the Relationship of Viral Particles and Extracellular Vesicles: Implications for Viral Vector Technology

“…When gaining information on single particles, ideally in a multi-parametric fashion, distinguishing, quantifying and sorting is possible. Indeed, a process very similar to flow cytometry, which has been termed flow virometry or nanovariant flow cytometry [ 57 ], is gaining more and more momentum. Furthermore, a combination of biological and physical parameters may help to unequivocally identify EVs and VIs.…”

Surface Modification of E. coli Outer Membrane Vesicles with Glycosylphosphatidylinositol-Anchored Proteins: Generating Pro/Eukaryote Chimera Constructs

“…For viral preparations, double painting has been described [36], however with the following serious drawback: the used method (immunoblotting) only gives averaged ensemble data (i.e., both proteins are found in the vesicle pool, no information is given about single vesicles). To rectify this situation, we established a nanovariant flow cytometry approach [8][9][10] to identify vesicles carrying both, or either/or, of the protein fluorophores (see Figures 5 and 6). 4) to show a significant number of events in the PBS or medium (M) controls.…”

“…This is a strategy that seems inherently reasonable, since OMVs, other EVs, and enveloped viral particles (e.g., lenti-, herpes-, influenza-or coronaviruses) have overlapping biophysical characteristics such as size and density [7]. Additionally, we tried to implement a flow cytometry approach adapted for sub-micron vesicles (also termed nanovariant flow cytometry nvFC, nanoscale flow cytometry NFC, or flow virometry) [8][9][10].…”

The P-selectin ligand PSGL-1 (CD162) is efficiently incorporated by primary HIV-1 isolates and can facilitate trans-infection

“…In our recent work we phenotyped pseudoviruses engineered to display the virionincorporated cellular proteins integrin α4β7, CD14 and PSGL-1 (CD162). This work led us to the striking observation that PSGL-1 was detected at markedly higher levels on virion surfaces compared to other host proteins, despite all pseudoviruses (PV) being produced with equal amounts of pDNA for host protein expression (28). Since we observed PSGL-1 to be incorporated to a greater extent than other host proteins on the surface of pseudoviruses, we wanted to determine how phenotypically similar pseudoviruses containing PSGL-1 were to viruses produced in more physiologically relevant model systems, such as infection of T cell of 48 lines and primary cells.…”

“…Flow virometry. Flow virometry was performed using a Beckman Coulter CytoFLEX S with standard optical configuration and volumetric calibrations were performed as described previously (28). Gain and threshold optimization for detection of virus and calibration beads was performed as described previously (68), with a modification of PE gain to 2600(68)(69).…”