Abstract
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.
Highlights
Convenient online submission Thorough peer review No space constraints or color figure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
We monitored the behavior of both membrane-bound and cytoplasmic HTLV-1 Gag in realtime in living cells incubated on a fluorescence microscope
We found that HTLV-1 Gag membrane-targeting occurred at all cytoplasmic concentrations measured, while appreciable membranetargeting for HIV-1 required Gag cytoplasmic concentration to exceed a threshold
Summary
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, Louis M Mansky1* From 15th International Conference on Human Retroviruses: HTLV and Related Viruses Leuven and Gembloux, Belgium.
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