A high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of HIV-1 viral membrane fusion

Anthony M. Esposito, Alexandra Y. Soare, Foramben Patel, Namita Satija, Benjamin K. Chen, Talia H. Swartz

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

Original languageEnglish
Article numbere58074
JournalJournal of Visualized Experiments
Volume2018
Issue number138
DOIs
StatePublished - 14 Aug 2018

Keywords

  • Cell-to-cell transmission
  • Cre recombinase
  • Entry
  • Fusion
  • HIV-1
  • Immunology and infection
  • Issue 138
  • Purinergic receptors
  • Screening

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