Comparing methods to detect cellular proteins on the surface of HIV-1 virions.

The surface of HIV-1 is embedded with numerous host-derived proteins. Characterizing these proteins can enhance knowledge of virus biology and potentially identify novel therapeutic targets. As many of these proteins are present in low abundance on virion surfaces, their identification can be hindered by inherent variables in the methods employed to detect them, including their varying assay sensitivities, sample processing, quantitative capacity, and experimental reproducibility. Here, we have compared the quantification of virion-incorporated proteins using conventional virus immunocapture assays and western blotting, alongside an emerging technique called flow virometry (FV). Using four different pseudovirus models that each express a human protein of interest (CD14, CD38, CD59 and CD162), we compared four experimental techniques for their ability to reliably quantify the incorporation of those four proteins onto virion surfaces. Our results shed light on the advantages and caveats of each technique for detecting virion-incorporated proteins and highlight the breadth in quantification for each technique under different experimental conditions. Protein detection with (FV) provided distinct advantages as it enabled highly reproducible quantifications, had the lowest sample requirements and reagent costs, and minimal hands-on experimental time. We additionally highlight some important considerations in experimental design when studying virion-incorporated proteins, such as the effect of different antibody clones, assay incubation times, and contributions of extracellular vesicles. Most importantly, our data illustrate the importance of using a combination of orthogonal approaches to detect virus-associated proteins, to enable reliable and reproducible quantification that accounts for individual assay biases.