The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient‐depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non‐enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long‐term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses. Two fundamental events in viral life cycles are the delivery of viral genomes into host cells and the packaging of these genomes into viral protein capsids. In bacteriophages and herpesviruses, these processes occur linearly along the genome, base pair after base pair, through a single portal located at a unique site in the viral capsid. We have addressed the question of whether such a linear translocation through a single portal also takes place for viruses harboring very large genomes, by studying genome delivery and packaging in the amoeba-infecting virus Acanthamoeba polyphaga mimivirus. With 1.2 million base pairs, this double-stranded DNA genome is the largest documented viral genome. By using electron tomography and cryo-scanning electron microscopy, we identified a large tunnel in the Mimivirus capsid that is formed shortly after infection, following a large-scale opening of the capsid. The tunnel allows the whole viral genome to exit in a rapid, one-step process. DNA encapsidation is mediated by a transient aperture in the capsid that, we suggest, may promote concomitant entry of multiple segments of the viral DNA molecule. These unprecedented modes of viral genome translocation imply that Mimivirus—and potentially other large viruses—evolved mechanisms that allow them to cope effectively with the exit and entry of particularly large genomes.

Poxviruses are considered to be unique among all DNA viruses, because their infection cycle is carried out exclusively in the host cytoplasm. Such an infection strategy is of interest, because it necessitates generation of elaborate factories in which viral replication and assembly are promoted. By using diverse imaging techniques, we show that the infection cycle of the largest virus currently identified, the Acanthamoeba polyphaga Mimivirus, similarly occurs exclusively in the host cytoplasm. We further show that newly synthesized mRNAs accumulate at discrete cytoplasmic sites that are distinct from the sites where viral replication occurs, and this is observed in vaccinia infection. By revealing substantial physiologic similarity between poxviruses and Mimivirus and thus, implying that an entirely cytoplasmic viral replication might be more common than generally considered, these findings underscore the ability of DNA viruses to generate large and elaborate replication factories.

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. With a particle size comparable to that of small bacteria and a 1.2 Mbp double-strand DNA genome that carries more than 1000 open reading frames, the amoeba-infecting Mimivirus, along with other recently identified members of the Mimiviridae family, are the largest and most complex viruses yet identified. The Mimivirus particle includes an internal membrane that underlies an icosahedral capsid. The assembly mechanism of internal membrane during Mimivirus infection remains unclear, as is the case for other viruses containing internal membranes. 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. This multistage process, which includes the formation of open membrane sheets, enables efficient and continuous assembly of multiple Mimivirus progeny. The membrane biogenesis process suggested here provides novel insights into the assembly of internal viral membranes in general.