We used cryo-electron tomography in conjunction with single-particle averaging techniques to study the structures of frozen-hydrated envelope glycoprotein (Env) complexes on intact Moloney murine leukemia retrovirus particles. Cryo-electron tomography allows 3D imaging of viruses in toto at a resolution sufficient to locate individual macromolecules, and local averaging of abundant complexes substantially improves the resolution. The averaging of repetitive features in electron tomograms is hampered by a low signal-to-noise ratio and anisotropic resolution, which results from the ''missingwedge'' effect. We developed an iterative 3D averaging algorithm that compensates for this effect and used it to determine the trimeric structure of Env to a resolution of 2.7 nm, at which individual domains can be resolved. Strikingly, the 3D reconstruction is shaped like a tripod in which the trimer penetrates the membrane at three distinct locations Ϸ4.5 nm apart from one another. The Env reconstruction allows tentative docking of the x-ray crystal structure of the receptorbinding domain. This study thus provides 3D structural information regarding the prefusion conformation of an intact unstained retrovirus surface protein.cryo-electron microscopy ͉ single-particle analysis ͉ Moloney murine leukemia virus T he surfaces of retroviruses are studded with a trimeric, type I transmembrane glycoprotein complex called ''Env.'' During infection, Env mediates binding of virus to its receptor in the cell plasma membrane and subsequent fusion of the viral and cellular membranes. Env undergoes large-scale conformational changes during these events (1, 2). Although the high-resolution structures of Env fragments from various retroviruses have been determined (3-9), the structure of intact Env on the virion surface is known only on a gross morphological level as a trimeric projection (10-12). The quaternary structure of Env on fresh virions is the starting point for the structural rearrangements that enable entry of the virus into cells and is therefore key to our understanding of the mechanism of retrovirus infection.Certain features of intact retroviral Env hamper its structural analysis by x-ray crystallography. For example, glycosylation, which can exceed 50% of the mass of Env, is an impediment to crystallization. Single-pass transmembrane proteins are notoriously difficult to crystallize; therefore attempts to replace the transmembrane region have been made by fusing the Env ectodomain to carrier proteins (13,14). Generation of a soluble Env ectodomain lacking transmembrane segments has typically involved removal of a proteolytic processing site (13), which prevents subunit dissociation but also prevents maturation of the complex into its final functional form. The large quantities of soluble Env necessary for crystallization attempts are difficult to prepare, and the material is often unstable and heterogeneous. Electron microscopy (EM) avoids many of these difficulties and provides useful structural information albeit at lower re...

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.

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.electron tomography | nucleocytoplasmic large DNA viruses | poxviruses | viral factories W ith the single exception of poxviruses, all currently known DNA viruses carry out replication and transcription either entirely or partially within host nuclei. The overwhelming bias to nucleus-centered viral transactions is rationalized by the fact that the nucleus provides the elaborate machinery required for these processes. Moreover, by concentrating essential factors into particular intranuclear sites as well as by enabling spatiotemporal regulation of viral replication and transcription, the nuclear environment considerably enhances the efficiency of these processes (1). Such underlying benefits render the entirely cytoplasmic infection cycle of poxviruses all of the more intriguing (2, 3). Nucleus-centered viral transactions present, however, remarkable hurdles associated with the prerequisite of transporting viral genomes from their entry sites at the cell periphery to and into the nucleus. Specifically, the cellular milieu is refractory to motion of DNA molecules because of the high viscosity of the cytosol and the dense molecular sieve generated by the cytoskeleton (4). Moreover, the nuclear envelope imposes a severe barrier for both entry and exit of long DNA molecules (1).The hurdles associated with genome trafficking are particularly high for nucleocytoplasmic large DNA viruses (NCLDV), which include the eukaryote-infecting families Poxviridae, Phycodnaviridae, Iridoviridae, and Asfarviridae, all characterized by DNA genomes longer than 100 kbp (5). After entry, genomes of phycodnaviruses (6), iridoviruses (7), and the African swine fever virus (8) are released into the cytoplasm at the host cell periphery and then, are shuttled toward and transported into the nucleus where initial replication cycles occur. Viral DNA is subsequently delivered to specific cytoplasmic factories in which all transactions required for viral assembly take place. The factors that attenuate the barriers imposed by the constrained DNA motion and thus, enabl...

Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.