Exploring the HTLV-1 Gag Membrane-Trafficking Pathway with Fluorescence Fluctuation Spectroscopy and Total Internal Reflection Fluorescence Microscopy

The use of biophysical fluorescence-based techniques for the study of proteins in living cells has significantly advanced in recent years. A particularly challenging aspect of fluorescence measurements conducted in live cells is in the study of protein systems which exhibit complex and varying behaviors in different regions of the cell. An example of such a system is retroviral Gag structural proteins, which play a prominent role in viral assembly and are known to have cytoplasmic and membrane-bound states with low-order and high-order oligomeric states, respectively. The presence of high-order, fluorescently-labeled Gag oligomers at the membrane can impact fluorescence monitoring of the cytoplasmic fraction of Gag. In addition, cytoplasmic species can confuse the study of membrane-bound Gag complexes. Our work focuses on the use of fluorescence fluctuation spectroscopy (FFS) and total internal reflection fluorescence (TIRF) microscopy to investigate retroviral Gag species, both at the membrane and in the cytoplasm. A focus of the study is the relationship between cytoplasmic Gag concentration and the formation of Gag assembly sites at the plasma membrane. FFS monitors Gag cytoplasmic concentration, while TIRF monitors the biogenesis of HTLV-1 virus-like particles. Comparison of results from HTLV-1 and HIV-1 Gag reveals intriguing differences in their assembly pathways. This work is supported by NIH Grant AI81673, NIH Grant GM064589, a Cancer Center Cancer Biology Training Grant (T32CA09138), and an American Cancer Society Postdoctoral Fellowship (PF-11-159-01-MPC).