Simultaneous Protein\nand RNA Analysis in Single Extracellular\nVesicles, Including Viruses

The individual detection of human immunodeficiency virus\n(HIV)\nvirions and resolution from extracellular vesicles (EVs) during analysis\nis a difficult challenge. Infectious enveloped virions and nonviral\nEVs are released simultaneously by HIV-infected host cells, in addition\nto hybrid viral EVs containing combinations of HIV and host components\nbut lacking replicative ability. Complicating the issue, EVs and enveloped\nvirions are both delimited by a lipid bilayer and share similar size\nand density. The feature that distinguishes infectious virions from\nhost and hybrid EVs is the HIV genomic RNA (gRNA), which allows the\nvirus to replicate. Single-particle analysis techniques, which provide\nsnapshots of single biological nanoparticles, could resolve infectious\nvirions from EVs. However, current single-particle analysis techniques\nfocus mainly on protein detection, which fail to resolve hybrid EVs\nfrom infectious virions. A method to simultaneously detect viral protein\nand internal gRNA in the same particle would allow resolution of infectious\nHIV from EVs and noninfectious virions. Here, we introduce SPIRFISH,\na high-throughput method for single-particle protein and RNA analysis,\ncombining single particle interferometric reflectance imaging sensor\nwith single-molecule fluorescence in situ hybridization. Using SPIRFISH,\nwe detect HIV-1 envelope protein gp120 and genomic RNA within single\ninfectious virions, allowing resolution against EV background and\nnoninfectious virions. We further show that SPIRFISH can be used to\ndetect specific RNAs within EVs. This may have major utility for EV\ntherapeutics, which are increasingly focused on EV-mediated RNA delivery.\nSPIRFISH should enable single particle analysis of a broad class of\nRNA-containing nanoparticles.