Novel Labeling Strategy for Automated Detection of Single Virus Fusion and Assessment of HIV-1 Protease Activity in Single Virions

Abstract

A common approach to visualizing single-virus fusion utilizes a fluorescently labeled Gag that, upon processing by HIV-1 viral protease, yields fluid-phase fluorescent proteins trapped within the virus particle. Upon loss of viral membrane integrity this content marker rapidly diffuses out of the particle, and, in combination with a viral core fiducial, such as a fluorescently tagged Vpr, can be used to reliably report single-particle fusion. But, this labeling approach is limited by an uncontrolled incorporation of the two markers, resulting in an excess of single-labeled particles and lack of correlation between intensities of the content and core markers. Here, we introduce a novel bi-functional mCherry-2xCL-YFP-Vpr label that encodes both the content (fluid-phase mCherry) and fiducial (core-associated YFP-Vpr) markers on a single construct separated by a 2xCL linker that contains a tandem of cleavage sites targeted by the viral protease. We show that the bi-functional label is efficiently incorporated and processed in single-virus particles and can be used to reliably report single-virus fusion in imaging assays. Further, by taking advantage of the nearly perfect colocalization and correlated intensities of content and fiducial markers in single-particles we are able to automate fusion event detection in both fixed and live cells, significantly increasing assay throughput. Finally, we show that the loss of Forster Resonance Energy Transfer (FRET) signal between YFP and mCherry is a good predictor of viral protease activity, and that the maturation status of single virions can be inferred by both FRET efficiency and YFP/mCherry signal ratio. Together, these results demonstrate that the new labeling strategy offers a number of advantages compared to the previous approaches, including reading out single-virus maturation status and increasing reliability and throughput of single-virus fusion imaging assays.