Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses

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