NEW INSIGHTS ON TRANSPORT AND PARTICLE DYNAMICS OBTAINED FROM REAL-TIME 3D TRACKING OF VIRAL PARTICLES IN LIVE CELLS

ANDRE O. GOMES, PHD1, ANDREA C. OLIVEIRA1, VALERIA LEVI1, RAFAEL B. GONCALVES2, UMESH KATPALLY3, THOMAS J. SMITH3, ENRICO GRATTON

Salt Lake City, Utah

Congreso; 50th annual meeting of the Biophysical Society; 2006

We investigated early events of virus infection in live cells by using the 3D tracking technique described by Levi et al. (Biophys J. 88: 2919-28, 2005). In this work, we followed Human Rhinovirus (HRV14) particles labeled with FITC entering into living HeLa-H1 cells, with nanometer accuracy and millisecond time resolution. We separated three different stages of the particle transport, specifically related to attachment to the cell surface, transport inside the cell and confinement in a region of the cell. Disruption of microfilaments and microtubules using latrunculin B and nocodazole, respectively, showed that particle transport in the cell is affected by actin depolimerization but not by tubulin breakdown. During the tracking experiment, the fluorescence intensity from the single particle is recorded and two interesting characteristics are shown: no photobleaching during tracking (>30 min) and an oscillatory intensity pattern, suggesting fast conformational changes of the particle’s size. Self-quenching due to FRET is expected in the highly labeled HRV14-Fluorescein conjugate. FLIM data revealed a very short lifetime for single particles inside cells, confirming very efficient FRET that could protect the conjugates from photobleaching. Any change in particle conformation that affects the efficiency of FRET is reflected in intensity fluctuations. Autocorrelation analyses of intensity data revealed that these fluctuations could be related to conformational dynamics of the HRV14 particle previously described as “breathing”. In addition, changes in the fluctuations characteristics and fluorescence lifetime were observed during infection, suggesting conformational transition of the particle that can be related to RNA release.Support: NIH - PHS P41 RR003155, CNPq/Brazil, FAPERJ, FUJB/UFRJ.