Volume 8 Supplement 1

15th International Conference on Human Retroviruses: HTLV and Related Viruses

Open Access

Analysis of the HTLV-1 Gag assembly pathway by biophysical fluorescence

  • Keir H Fogarty1, 2,
  • Yan Chen2,
  • Iwen F Grigsby1,
  • Patrick J Macdonald2,
  • Elizabeth M Smith2,
  • Jolene L Johnson2,
  • Jonathan M Rawson1,
  • Joachim D Mueller1, 2 and
  • Louis M Mansky1Email author
Retrovirology20118(Suppl 1):A206

DOI: 10.1186/1742-4690-8-S1-A206

Published: 6 June 2011

Much of the mechanistic details for how HTLV-1 Gag orchestrates virus particle assembly and release are poorly understood. Here, we monitored the behavior of both membrane-bound and cytoplasmic HTLV-1 Gag in real-time in living cells incubated on a fluorescence microscope. We used both fluorescence fluctuation spectroscopy (FFS, conventional and z-scan) and fluorescence imaging (epi-illumination, total internal reflection fluorescence (TIRF)) to investigate the relationship between cytoplasmic and membrane bound Gag, using a Gag-YFP model system. FFS determines the brightness, mobility, and concentration (conventional) and localization (z-scan) of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume, which yields information about protein stoichiometry, interactions, transport, and distribution. By coupling the single-molecule FFS technique with imaging techniques capable of monitoring Gag localization (epi-illumination) and membrane-specific localization (TIRF), we achieved new insights into the earliest events in HTLV-1 Gag assembly, and differences to HIV-1 Gag. We found that HTLV-1 Gag membrane-targeting occurred at all cytoplasmic concentrations measured, while appreciable membrane-targeting for HIV-1 required Gag cytoplasmic concentration to exceed a threshold. In addition, z-scan FFS revealed that a substantial population of membrane-bound HTLV-1 Gag exists not as puncta, but as a diffuse, low-order, dynamic “sheet.” These observations, coupled with previous observations of cytoplasmic Gag interactions and mobility, point to differences in membrane targeting of HTLV-1 and HIV-1 Gag. In summary, the suite of biophysical fluorescence techniques, applied HTLV-1 Gag, provide unparalleled information concerning HTLV-1 Gag trafficking processes in vivo, elucidating assembly pathway differences between HTLV-1 and other retroviruses.

Authors’ Affiliations

(1)
Institute for Molecular Virology, University of Minnesota
(2)
School of Physics and Astronomy, University of Minnesota

Copyright

© Fogarty et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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