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Author Notes:

To whom correspondence should be addressed. Tel.: 404-727-4652; E-mail: gmeliki@emory.edu

C.S. and A.C.F. contributed equally to this work.

A. C. F. created and optimized the bifunctional marker.

C. S., A. C. F., and G. B. M. conceived and planned the experiments.

C. S., A. C. F., and T. M. D. performed the experiments and analyzed the results.

C. S. wrote software for automated detection of fusion.

C. S., A. C. F., and G. B. M. wrote the manuscript.

All authors read and approved the manuscript.

We thank Mariana Marin for help with Western blotting, critical reading of the manuscript, and helpful discussions.

We thank the National Institutes of Health AIDS Reagent Program, David Kabat, and James Binley for cell lines and plasmids and Dr. E. Gratton for valuable suggestions regarding virus fusion analysis.

The authors declare that they have no conflicts of interest with the contents of this article.

Subjects:

Research Funding:

This work was supported by National Institutes of Health Grants R01 GM054787 and R01 AI053668 (to G. B. M.).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • confocal microscopy
  • FRET
  • retrovirus
  • single particle analysis
  • virus entry
  • HIV maturation
  • live-cell imaging
  • fusion pore
  • MEMBRANE-FUSION
  • PARTICLE FUSION
  • PORE FORMATION
  • LIVING CELLS
  • HEMIFUSION
  • MATURATION
  • VISUALIZATION
  • GLYCOPROTEIN
  • ENDOCYTOSIS
  • ENDOSOMES

An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions

Journal Title:

Journal of Biological Chemistry

Volume:

Volume 292, Number 49

Publisher:

, Pages 20196-20207

Type of Work:

Article | Final Publisher PDF

Abstract:

Enveloped viruses transfer their genomes into host cells by fusing their membrane to that of the cell. To visualize singlevirus fusion in living cells, researchers take advantage of the proteolytic maturation of HIV, type 1 (HIV-1), which can generate free fluorescent proteins within the viral particle. Co-labeling viruses with a content marker and a fluorescently tagged Vpr (a viral core protein) enables detection of single-virus fusions, but a major limitation of this approach is that not all viral particles incorporate both markers. Here we designed a labeling strategy based on the bifunctional mCherry-2xCLYFP- Vpr construct, in which 2xCL denotes a tandem cleavage site for the viral protease. This bifunctional marker was efficiently cleaved during virus maturation, producing free mCherry and the core-associated YFP-Vpr. A nearly perfect colocalization of these two markers in virions and their fixed 1:1 ratio enabled automated detection of single-particle fusion in both fixed and live cells based on loss of the mCherry signal. Furthermore, a drop in FRET efficiency between YFP and mCherry because of cleavage of the bifunctional marker, which manifested as a marked shift in the normalized YFP/mCherry fluorescence ratio, reliably predicted viral protease activity in single virions. This feature could discriminate between the particles containing free mCherry, and therefore likely representing mature viruses, and immature particles whose fusion cannot be detected. In summary, our new labeling strategy offers several advantages compared with previous approaches, including increased reliability and throughput of detection of viral fusion. We anticipate that our method will have significant utility for studying viral fusion and maturation.

Copyright information:

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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