Isolation of an Asymmetric RNA Uncoating Intermediate for a Single-Stranded RNA Plant Virus

We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ra...

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Veröffentlicht in:Journal of molecular biology 2012-03, Vol.417 (1-2), p.65-78
Hauptverfasser: Bakker, Saskia E., Ford, Robert J., Barker, Amy M., Robottom, Janice, Saunders, Keith, Pearson, Arwen R., Ranson, Neil A., Stockley, Peter G.
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Sprache:eng
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Zusammenfassung:We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity. [Display omitted] ► Native and expanded TCV cryo-electron microscopy structures reveal RNA dynamics. ► Expansion leads to the formation of pores large enough to allow exit of viral RNA. ► RNA remains resistant to nuclease, but capsid proteins become protease sensitive. ► Proteolysis causes initial extrusion of viral RNA, creating asymmetry in capsids. ► Subsequent ribosome binding prevents silencing and promotes genome extrusion.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2012.01.017