Cryo-electron tomography of phi12 bacteriophage

The bacteriophage phi12 is a member of the family Cystoviridae, a unique group of lipid-containing, membrane-enveloped bacteriophages that infect strains of the plant bacterial pathogen Pseudomonas syringae pv. phaseolicola. These bacteria are what commonly cause lettuce and other leafy vegetables to decay. This Cystoviridae family of viruses is characterized by a segmented double-stranded RNA genome, divided into three segments and named according to their size: small (S), medium (M), and large (L). Members of Cystoviridae have been very useful research models for elucidating replication mechanisms of several RNA viruses.

The inner-most virion layers have isocahedral organization and are similar at the structural level, indicating conserved mechanisms of genome packaging, replication and transcription. These icosahedral parts of the virus have been studied by cryo-electron microscopy using standard icosahedral reconstruction techniques. In contrast, the outer virion layer mediates host interactions and has diverged to assist recognition and entry into a variety of host cells, thus extending the host range. This outer layer comprises a lipid bilayer and randomly arranged membrane proteins and must be addressed by electron tomography methods.

phi12 organization

 

Members of Cystoviridae that have been studied by electron microscopy show characteristics "spikes", that correspond to the protein complex responsible for host recognition and entry. In cryo-electron tomograpms, we found that these spikes are actually toroidal protrusions. Since they do not conform to the icosahedral symmetry of the NC we study their shape and distribution on the surface of the virus by cryo-electron tomography.

Image on the right represents a surface rendering of a single virion. False color has been used to illustrate the presence of two types of surface projections: toroidal (red) and elongated (blue).

phi12 virion

slices through phi12 tomogram

(A) A slice from a cryo-electron tomogram with a blow-up of a single virion in the inset. (B) A series of slices through a single virion showing a number of surface projections that have a toroidal shape.

By using sub-tomogram averaging, we have improved the resolution of this toroidal projection dramatically. After averaging 744 copies of the toroidal surface projection extracted from 158 virions, we have obtained a structure with ~25A resolution. This structure shows that the toroidal complex has six-fold symmetry, and is connected to the virus envelope surface by a discrete density. Furthermore, a genetic reassortant of phi12 has been used to demonstrate unambiguously that this hexameric surface element is responsible for host specificity . Our findings have important implications in regard to the mechanisms of viral evolution and their adaptation to additional host cells. The receptor binding protein, P3 is the viral component usually altered in response to host change. Future work will focus on the structure of P3 and its relationship to host cell binding in different species and strains of cystoviruses such as phi13.

Subtomogram averaging of phi12

(A-C) Sections through the averaged donut along three orthogonal axes. (D-F) Surface renderings of the averaged structure showing dimensions of the various elements. (G) Synthetic rendering of the surface of a virion with both toroidal and elongated surface projections.