A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry
Text by Anthony Esposito
While most drug development for HIV has focused on virus infection in which HIV is released from cells, it has been shown that HIV is capable of infecting new cells directly through cell-cell contacts known as virological synapses. This direct mode of cell-to-cell transmission has been shown to be more resistant to specific antiretrovirals such as tenofovir. For this reason it is important to consider cell-to-cell transmission when developing novel HIV inhibitors. To this end we developed an assay that would allow us to test potential inhibitors rapidly with a red to green fluorescent color changing reporter system which utilizes a modified HIV that packages a DNA recombinase enzyme Cre, called Gag-iCre. Inhibitors of viral entry prevent the Cre-induced color change which can easily be detected by a color change. We used the assay to screen 1998 compounds, identifying some novel inhibitors and confirming some previously identified inhibitors.
Our primary motivation in designing a new assay to measure viral entry came from our interest in high thoughput screens in a cell-to-cell transmission system. A current viral membrane fusion assay uses the delivery of an enzyme, beta lactamase, to a target cell by fusing the beta lactamase gene to the viral protein vpr. While an excellent assay for some purposes, the high cost of the required substrate limits its use in larger screens. We attempted many variants involving luciferase based reporters as well as protein complementation. Eventually we thought of testing a cre recombinase based reporter and realized it would suit a fusion assay very well if the recombinase could be packaged into particles. We decided to try fusing cre to the most abundant protein in the virus particle Gag in a position where we had prior success inserting other molecules, such as GFP. The assay was then developed and used to perform the screen described in the paper. Using high throughput flow cytometry we were able to get through many compounds with the assay in its current form, we realized for larger compound libraries. The assay provides a nice alternative to the use of beta lactamase cells.
Overview of Cre-mediated viral membrane fusion assay. The cre recombinase gene was inserted into the Gag polyprotein as an internal fusion protein between the matrix and capsid domains and flanked by protease cleavage sites. This allows Cre recombinase to be packaged into virus particles and processed by the viral protease to yield particles that contain enzymatically active, Cre. These virus used to infect target cell lines containing a floxed dsRed to GFP expression cassette. When viral membrane fuses with the target cell membrane, Cre is released into target cells and recombines out the dsRed resulting in the GFP being expressed that is directly proportional to the amount of fusion. Inhibition of GFP is measured to identify inhibitors of HIV membrane fusion.
Introducing the authors
From left to right: Benjamin Chen, Talia Swartz, Anthony Esposito, Natasha Durham and Hongru Li.
About the research
A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry
Anthony M. Esposito, Pamela Cheung, Talia H. Swartz, Hongru Li, Tshidi Tsibane, Natasha D. Durham, Christopher F. Basler, Dan P. Felsenfeld, Benjamin K. Chen
Virology, Volume 490, March 2016, Pages 6–16