Fluorescence Microscopy Journal Club

Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

by Alonas et al.

ACS Nano, 2014, 8, 1, pp. 302–315

Super-resolution imaging has been gaining popularity and use in recent research. This week’s article shows an example of how super-resolution imaging can be used to study the distribution of viral matrix protein (M) and cellular proteins such as F-actin using newly developed labeling techniques, and can be utilized as general methodology for studying RNA viruses such as influenza and Ebola.

This paper presents a novel method for fluorescently labeling genomic RNA (gRNA) of hSRV (human respiratory syncytial virus) virions using multiply labeled tetravalent RNA imaging probes (MTRIPs).

Cells were infected with hRSV and viral RNA was labeled using MTRIPs. The virions were then harvested from the cell, deposited on glass coverslips and subsequently stained for either the viral nucleoprotein (N) or viral fusion protein (F). Filamentous virion morphology was characterized using three dimensional single molecule localization super-resolution microscopy. Super-resolution images showed that the F-protein was distributed evenly along the length of the filaments in contrast to gRNA and N protein which were distributed unevenly. The gRNA and N protein overlapped tightly in co-localized areas whereas the F protein appeared to surround the gRNA. The axial distributions indicated that F protein was located away from the center of the virion when compared to N protein and gRNA.