Date Published: May 30, 2013
Publisher: Public Library of Science
Author(s): Yael Mutsafi, Eyal Shimoni, Amir Shimon, Abraham Minsky, Raul Andino.
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.
Nucleocytoplasmic large DNA viruses (NCLDVs), which include Poxviridae, Phycodnaviridae, Iridoviridae, Asfarviridae and Mimiviridae, 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 –. A critical process occurring in these factories is the assembly of inner viral membranes that are present in all NCLDVs. The origin of these membranes, their assembly as well as their number within NCLDV virions remain poorly understood.
In large DNA viruses the origin of membrane layers, their numbers, as well as the mechanisms responsible for their assembly remain controversial , , –. The current leading view is that these viruses carry a single inner membrane bilayer , , , , raising the question how is such a single bilayer generated. Specifically, whereas formation of two membranes can be straightforwardly assigned to wrapping of collapsed ER cisternae, a single bilayer requires either single-layer membrane precursors or the loss of one ER membrane layer by yet unidentified mechanisms. Insights into these questions were recently provided by cryo-EM and electron tomography of Vaccinia-infected cells , which revealed that Vaccinia crescent precursors are single membrane sheets generated through rupture of the host ER membrane network.