Extracellular uncoating of bacteriophage MS2

Amelia W Paine; Rees F Garmann; Vinothan N Manoharan

doi:10.1016/j.jmb.2025.169316

Extracellular uncoating of bacteriophage MS2

J Mol Biol. 2025 Jul 2:169316. doi: 10.1016/j.jmb.2025.169316. Online ahead of print.

Authors

Amelia W Paine¹, Rees F Garmann², Vinothan N Manoharan³

Affiliations

¹ Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
² Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182, USA; Viral Information Institute, San Diego State University, San Diego, CA 92182, USA. Electronic address: rgarmann@sdsu.edu.
³ Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA; Department of Physics, Harvard University, Cambridge, MA 02138, USA. Electronic address: vnm@seas.harvard.edu.

PMID: 40614858
DOI: 10.1016/j.jmb.2025.169316

Abstract

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.