Abstract
Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, ∼70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general.
Highlights
Nucleocytoplasmic large DNA viruses (NCLDVs), which include Poxviridae, Phycodnaviridae, Iridoviridae, Asfarviridae and Mimiviridae [1], replicate and assemble in cytoplasmic inclusions called viral factories
By using diverse imaging techniques, we showed that membrane biogenesis is an elaborate process that occurs at the periphery of viral factories generated at the host cytoplasm
Since our studies of membrane biogenesis during Mimivirus infection were carried out using mainly Scanning-Transmission Electron Microscopy (STEM) tomography, we analyzed the structure of mature intracellular Mimivirus virions using the same technique in order to enable direct comparison (Fig. 1A–C; Movie S1)
Summary
Nucleocytoplasmic large DNA viruses (NCLDVs), which include Poxviridae, Phycodnaviridae, Iridoviridae, Asfarviridae and Mimiviridae [1], replicate and assemble in cytoplasmic inclusions called viral factories Formation of these elaborate structures that enable spatial and temporal coordination of viral assembly and effective recruitment of host factors, involves massive rearrangement of host cytoskeleton and membranes [2,3,4,5]. A single membrane envelope is commonly acquired by viral budding into intracellular compartments or through plasma membranes Since such processes were not observed during the assembly of IV particles, and as the initial viral membrane structures, dubbed crescents, did not demonstrate a continuity with host membranes, it was proposed that such crescents are generated by a de novo synthesis from lipid precursors and have open ends in the cytoplasm [6]. The two-membrane model was challenged by freeze-fracture electron microscopy studies, which demonstrated that IV membrane consists of a single bilayer stabilized by a protein coat [9,14]
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