Yael Mutsafi

Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus.

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.

Vaccinia-like cytoplasmic replication of the giant Mimivirus

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.

Membrane assembly during the infection cycle of the giant Mimivirus.

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.

Virus–host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus

The increasing interest in cytoplasmic factories generated by eukaryotic‐infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV‐1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV‐1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV‐1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane‐containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV‐1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage‐like infection pathway.

3D visualization of mitochondrial solid-phase calcium stores in whole cells

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

ABSTRACT The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed “viral factories,” where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as “production lines” of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed “viral factories,” are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.

cis-Proline Mutants of Quiescin Sulfhydryl Oxidase 1 With Altered Redox Properties Undermine ECM Integrity and Cell Adhesion in Fibroblast Cultures: QSOX1 cis-proline mutants

The thioredoxin superfamily has expanded and diverged extensively throughout evolution such that distant members no longer show appreciable sequence homology. Nevertheless, redox‐active thioredoxin‐fold proteins functioning in diverse physiological contexts often share canonical amino acids near the active‐site (di‐)cysteine motif. Quiescin sulfhydryl oxidase 1 (QSOX1), a catalyst of disulfide bond formation secreted by fibroblasts, is a multi‐domain thioredoxin superfamily enzyme with certain similarities to the protein disulfide isomerase (PDI) enzymes. Among other potential functions, QSOX1 supports extracellular matrix assembly in fibroblast cultures. We introduced mutations at a cis‐proline in QSOX1 that is conserved across the thioredoxin superfamily and was previously observed to modulate redox interactions of the bacterial enzyme DsbA. The resulting QSOX1 variants showed a striking detrimental effect when added exogenously to fibroblasts: they severely disrupted the extracellular matrix and cell adhesion, even in the presence of naturally secreted, wild‐type QSOX1. The specificity of this phenomenon for particular QSOX1 mutants inspired an investigation of the effects of mutation on catalytic and redox properties. For a series of QSOX1 mutants, the detrimental effect correlated with the redox potential of the first redox‐active site, and an X‐ray crystal structure of one of the mutants revealed the reorganization of the cis‐proline loop caused by the mutations. Due to the conservation of the mutated residues across the PDI family and beyond, insights obtained in this study may be broadly applicable to a variety of physiologically important redox‐active enzymes.

Amorphous Solid Phase Deposition of Ions and Phosphate within Eukaryotic Mitochondrial Matrices - Imaging and Characterization by CryoSTEM Tomography and Energy- Dispersive X-ray Spectroscopy

CryoSTEM tomography (CSTET) is a novel combination of two well-established techniques: cryoelectron tomography (CET) and scanning transmission electron microscopy (STEM). The best current method for faithful preservation of cells is provided by cryogenic fixation. CET of such cryo-preserved prokaryotic and eukaryotic cells has provided rich detail on the in-situ organization of macromolecules and organelles. However, sample thickness is limited to around 300 nm (close to the mean free path for inelastic scattering), due to the dependence on phase contrast from elastically scattered electrons. Using energy filters, inelastically scattered electrons can be blocked off, but this reduces signal at higher tilts while still depositing damaging energy into the sample. In addition, correction of the contrast transfer function at high tilts is not straightforward.

202 Structural aspects of the infection cycles of giant viruses

With a particle size of ∼800 nm and a DNA genome of 1.2 M base-pairs, the recently discovered amoeba-infecting virus Mimivirus is the largest virus identified, blurring the established division between viruses and single-cell organisms. Such unusual parameters raise fundamental questions related to the physical aspects of the Mimivirus infection cycle. These include the mechanisms that promote entry of the huge Mimivirus genome into host cells and its trafficking within the highly crowded host cytoplasm, virion assembly, and genome packaging. Our studies indicate that, in contrast to all other DNA viruses, the Mimivirus releases its genome into the host cytoplasm in a single step that is promoted by a large-scale conformational change of the viral capsid (Figure). This process is followed by the assembly of a large and highly elaborate viral factory in the host cytoplasm within which multiple viral progeny are rapidly generated in a pathway that is independent of the host nucleus (Zauberman et al., 2008). The transactions that occur in the viral factory, including replication, transcription, translation, membrane biogenesis, and capsid assembly are well-coordinated in time and space (Mutsafi et al., 2010; Mutsafi et al., submitted), thus, providing an unusual and exciting case study in self-assembly. Moreover, the transactions that lead to the generation of viral factories raise the intriguing notion that such factories might have acted as precursors to eukaryotic nuclei. The implications of these findings on the evolution of viruses and the role viruses might have played in the emergence of eukaryotic cells will be discussed.