Technical Highlight - March 2014
Short description: The first application of ZPC cryoET provides insight into cyanophage assembly process.
Cyanophages are double-stranded DNA viruses that infect a wide range of photosynthetic cyanobacteria. Using a new electron microscopy approach, Zernike phase contrast electron cryo-tomography (ZPC cryoET), Chiu and colleagues analyzed the assembly pathway of the cyanophage Syn5 in its natural host cells at nanometer resolution.
Synechococcus sp. strain WH8109 cells were frozen before and after infection, and imaged in an electron microscope equipped with a Zernike phase plate placed in the back focal plane of the objective lens. This substantially enhanced image contrast compared to conventional cryoET. The authors reconstructed 58 ZPC tomograms of the cells and classified 470 intracellular Syn5-like particles into three morphological types, which were further analyzed to obtain 50–70-Å resolution averages.
The largest population of particles resembles mature Syn5 phage, a short-tailed icosahedral capsid shell with a horn appendage at the vertex opposite the tail and an internal density that is attributed to DNA. Three particle subtypes were observed: those with a bulky tail and a slim horn appendage on opposing vertices, as in the mature phage; a tail at one vertex only; and without detectable density protruding from any vertex. This categorization suggests that the assembly of the tail hub follows DNA packaging, but precedes the addition of the horn.
The second phage population, thought to represent Syn5 procapsids, consists of spherical particles that are slightly smaller than mature Syn5, with a density extending inward at one location of the shell, which is interpreted to represent the Syn5 portal. The third particle type, expanded capsids, had not been previously reported and is similar to mature Syn5 in size and angular in shape, with the majority of particles displaying icosahedral symmetry. An internally protruding density is suggested to correspond to the full-length portal protein complex, whereas an external protruding density might represent the terminase.
By mapping the frequency of the three phage populations as infection progresses, procapsids and expanded capsids appear first, but disappear again as the population of DNA-containing capsids grows. Thus, the expanded capsids represent an intermediate state in the assembly process, and expansion and angularity of the protein shell are completed before the viral DNA is fully packaged. This morphogenetic assembly pathway resembles that of enteric bacteriophages and eukaryotic viruses, suggesting that it is highly conserved and likely inherited from cyanobacteria, which precede enteric bacteria in evolution.
W. Dai et al. Visualizing virus assembly intermediates inside marine cyanobacteria.
Nature. 502, 707-710 (2014). doi:10.1038/nature12604