Extracellular uncoating of bacteriophage MS2.
In the early stages of infection of its host, Escherichia coli, bacteriophage MS2 sheds its icosahedral protein capsid, after which the single-stranded genomic RNA (gRNA) and maturation protein enter the cell as a complex. Although the steps preceding uncoating, which include the binding of the Mat protein to the extracellular filament F-pilus, have been studied in detail, the uncoating step is not well understood. To study when and where uncoating happens, we image the infection process using fluorescence microscopy, separately labelling the MS2 capsid, its gRNA, and the cells. We do two types of experiments. In the first, we incubate the phage in a nonspecific intercalating dye, and we count the number of uncoated and intact phages before and after adding the labeled phages to cells. In the second, we examine the time course of infection by fixing unlabeled samples at different times after adding the phage, and then we label the MS2 gRNA using amplified fluorescence in situ hybridization. In both cases, we find that uncoating can occur anywhere on the F-pili, and that MS2 usually uncoats at a distance from the cell rather than at the cell surface. While these results do not rule out a current hypothesis that virus particles uncoat when the F-pilus retracts and brings them into contact with the cell body, they demonstrate an alternative, extracellular uncoating pathway. We discuss the possiblity that MS2 may have multiple uncoating pathways, and that the rate of each pathway could reflect a trade-off between different risk factors.