Visualization of viral assembly in the infected cell.

The study of the virus life cycle in infected cells is a methodological challenge due to the small size and diversity of the viral components. Recent developments on preservation of fine structure and molecular localization have provided a group of powerful methods with wide applications in cell biology and virology. Among the different electron microscopy (EM) techniques available to visualize viral assembly at the intracellular level, we will focus on conventional ultrathin sections, cryosections, and freeze-substitution. For obtaining molecular information associated to ultrastructure we have now a group of methods to detect viral proteins (immunogold labeling), as well as the viral genome, through the different techniques for detection of nucleic acids (the enzyme-gold approach, in situ hybridization, and elemental mapping). We will illustrate the applications of these methods with examples of viruses that exhibit different levels of structural complexity. These new approaches help to detect and identify viruses in clinical samples and to characterize the virus life cycle and the cellular components involved, to obtain data that could help for a therapeutic intervention, and to characterize virus-like particles that can be the basis of new and safe vaccines.     

Structure of complex viruses and virus-infected cells by electron cryo tomography

In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail.     

Parvovirus replication.

The members of the family Parvoviridae are among the smallest of the DNA viruses, with a linear single-stranded genome of about 5 kilobases. Currently the family is divided into three genera, two of which contain viruses of vertebrates and a third containing insect viruses. This review concentrates on the vertebrate viruses, with emphasis on recent advances in our insights into the molecular biology of viral replication. Traditionally the vertebrate viruses have been distinguished by the presence or absence of a requirement for a coinfection with a helper virus before productive infection can occur, hence the notion that the dependoviruses (adeno-associated viruses [AAV]) are defective. Recent data would suggest that not only is there a great deal of structural and genetic organizational similarity between the two types of vertebrate viruses, but also there is significant similarity in the molecular biology of productive replication. What differs is the physiological condition of the host cell that renders it permissive. Healthy dividing cells are permissive for productive replication by autonomous parvoviruses; such cells result in latent infection by dependoviruses. For a cell to become permissive for productive AAV replication, it must have been exposed to toxic conditions which activate a latent AAV genome. Such conditions can be caused by helper-virus infection or exposure to physical (UV light) or chemical (some carcinogens) agents. In this paper the molecular biology of replication is reviewed, with special emphasis on the role of the host and the consequences of viral infection for the host.     

Parvovirus replication.

The members of the family Parvoviridae are among the smallest of the DNA viruses, with a linear single-stranded genome of about 5 kilobases. Currently the family is divided into three genera, two of which contain viruses of vertebrates and a third containing insect viruses. This review concentrates on the vertebrate viruses, with emphasis on recent advances in our insights into the molecular biology of viral replication. Traditionally the vertebrate viruses have been distinguished by the presence or absence of a requirement for a coinfection with a helper virus before productive infection can occur, hence the notion that the dependoviruses (adeno-associated viruses [AAV]) are defective. Recent data would suggest that not only is there a great deal of structural and genetic organizational similarity between the two types of vertebrate viruses, but also there is significant similarity in the molecular biology of productive replication. What differs is the physiological condition of the host cell that renders it permissive. Healthy dividing cells are permissive for productive replication by autonomous parvoviruses; such cells result in latent infection by dependoviruses. For a cell to become permissive for productive AAV replication, it must have been exposed to toxic conditions which activate a latent AAV genome. Such conditions can be caused by helper-virus infection or exposure to physical (UV light) or chemical (some carcinogens) agents. In this paper the molecular biology of replication is reviewed, with special emphasis on the role of the host and the consequences of viral infection for the host.     

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.     

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.     

富含半胱氨酸病毒蛋白的研究进展

病毒编码的富含半胱氨酸的小分子量蛋白(CRPs)在植物和动物病毒中均有发现。动物病毒中研究较多的是反转录病毒的核蛋白(NC)。在植物病毒中由hordei,tobra,furoandcarlaviruses编码的CRPs的分子生物学研究近年来才开展起来。动物和植物病毒的CRPs共有的典型特征是均有锌指结构和碱性氨基酸丰富区,这使它们在核酸结合特性上有共同特征。动物病毒CRPs的结构和功能方面的研究已有很好的进展。相反,植物病毒的CRPs的研究进展较为缓慢。本文对病毒的CRPs的最新进展进行了综述。对动物和植物病毒的CRPs的比较分析有助于将来这类蛋白功能的阐明。     

Virus factories: biogenesis and structural design

Replication and assembly of many viruses occur in specific intracellular compartments known as ‘virus factories’. Our knowledge of the biogenesis and architecture of these unique structures has increased considerably in the last 10 years, due to technical advances in cellular, molecular and structural biology. We now know that viruses build replication organelles, which recruit cell and viral components in a macrostructure in which viruses assemble and mature. Cell membranes and cytoskeleton participate in the biogenesis of these scaffolds and mitochondria are present in many factories, where they might supply energy and other essential factors. New inter‐organelle contacts have been visualized within virus factories, whose structure is very dynamic, as it changes over time. There is increasing interest in identifying the factors involved in their biogenesis and functional architecture, and new microscopy techniques are helping us to understand how these complex entities are built and work. In this review, we summarize recent findings on the cell biology, biogenesis and structure of virus factories.     

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.     

Viruses in Biology

During the first half of the twentieth century, many scientists considered viruses the smallest living entities and primitive life forms somehow placed between the inert world and highly evolved cells. The development of molecular biology in the second half of the century showed that viruses are strict molecular parasites of cells, putting an end to previous virocentric debates that gave viruses a primeval role in the origin of life. Recent advances in comparative genomics and metagenomics have uncovered a vast viral diversity and have shown that viruses are active regulators of cell populations and that they can influence cell evolution by acting as vectors for gene transfer among cells. They have also fostered a revival of old virocentric ideas. These ideas are heterogeneous, extending from proposals that consider viruses functionally as living beings and/or as descendants of viral lineages that preceded cell evolution to other claims that consider viruses and/or some viral families a fourth domain of life. In this article, we revisit these virocentric ideas and analyze the place of viruses in biology in light of the long-standing dichotomic debate between metabolist and geneticist views which hold, respectively, that self-maintenance (metabolism) or self-replication and evolution are the primeval features of life. We argue that whereas the epistemological discussion about whether viruses are alive or not and whether some virus-like replicators precede the first cells is a matter of debate that can be understood within the metabolism-versus-genes dialectic; the claim that viruses form a fourth domain in the tree of life can be solidly refuted by proper molecular phylogenetic analyses and needs to be removed from this debate.     

Electron tomography of cells

The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or 'tomogram'. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.     

Whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction.     

Endogenous viruses: insights into viral evolution and impact on host biology

Recent studies have uncovered myriad viral sequences that are integrated or 'endogenized' in the genomes of various eukaryotes. Surprisingly, it appears that not just retroviruses but almost all types of viruses can become endogenous. We review how these genomic 'fossils' offer fresh insights into the origin, evolutionary dynamics and structural evolution of viruses, which are giving rise to the burgeoning field of palaeovirology. We also examine the multitude of ways through which endogenous viruses have influenced, for better or worse, the biology of their hosts. We argue that the conflict between hosts and viruses has led to the invention and diversification of molecular arsenals, which, in turn, promote the cellular co-option of endogenous viruses.     

Vaccinia virus A11 is required for membrane rupture and viral membrane assembly

Although most enveloped viruses acquire their membrane from the host by budding or by a wrapping process, collective data argue that nucleocytoplasmic large DNA viruses (NCLDVs) may be an exception. The prototype member of NCLDVs, vaccinia virus (VACV) may induce rupture of endoplasmic‐reticulum‐derived membranes to build an open‐membrane sphere that closes after DNA uptake. This unconventional membrane assembly pathway is also used by at least 3 other members of the NCLDVs. In this study, we identify the VACV gene product of A11, as required for membrane rupture, hence for VACV membrane assembly and virion formation. By electron tomography, in the absence of A11, the site of assembly formed by the viral scaffold protein D13 is surrounded by endoplasmic reticulum cisternae that are closed. We use scanning transmission electron microscopy–electron tomography to analyse large volumes of cells and demonstrate that in the absence of A11, no open membranes are detected. Given the pivotal role of D13 in initiating VACV membrane assembly, we also analyse viral membranes in the absence of D13 synthesis and show that this protein is not required for rupture. Finally, consistent with a role in rupture, we show that during wild‐type infection, A11 localises predominantly to the small ruptured membranes, the precursors of VACV membrane assembly. These data provide strong evidence in favour of the unusual membrane biogenesis of VACV and are an important step towards understanding its molecular mechanism.     

Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.     

嗜神经性疱疹病毒：神经科学研究的有力工具

病毒是研究现代神经科学的有力工具。对神经元的连接方式及功能研究大都是利用重组病毒完成的,嗜神经性疱疹病毒便是其中一种重要工具。随着基因工程学以及分子生物学技术的不断发展,多种嗜神经性疱疹病毒被改造为不同的重组病毒工具应用于神经科学研究。本文基于几种常见且应用较为广泛的嗜神经性疱疹病毒作为神经传导示踪工具、治疗神经性疾病的病毒载体和溶瘤病毒治疗神经肿瘤等应用进行阐述及讨论,为进一步开发嗜神经性疱疹病毒的功能提供参考。     

G-quadruplexes in genomes of viruses infecting eukaryotes or prokaryotes are under different selection pressures from hosts

G-quadruplexes in viral genomes can be applied as the targets of antiviral therapies, which has attracted wide interest. However, it is still not clear whether the pervasive number of such elements in the viral world is the result of natural selection for functionality. In this study, we identified putative quadruplex-forming sequences (PQSs) across the known viral genomes and analyzed the abundance, structural stability, and conservation of viral PQSs. A Viral Putative G-quadruplex Database (http://jsjds.hzau.edu.cn/MBPC/ViPGD/index.php/home/index) was constructed to collect the details of each viral PQS, which provides guidance for selecting the desirable PQS. The PQS with two putative G-tetrads (G₂-PQS) was significantly enriched in both eukaryotic viruses and prokaryotic viruses, whereas the PQSs with three putative G-tetrads (G₃-PQS) were only enriched in eukaryotic viruses and depleted in prokaryotic viruses. The structural stability of PQSs in prokaryotic viruses was significantly lower than that in eukaryotic viruses. Conservation analysis showed that the G₂-PQS, instead of G₃-PQS, was highly conserved within the genus. This suggested that the G₂-quadruplex might play an important role in viral biology, and the difference in the occurrence of G-quadruplex between eukaryotic viruses and prokaryotic viruses may result from the different selection pressures from hosts.     

Electron microscope tomography

Electron microscope tomography allows three-dimensional reconstruction of ultrastructural objects at the molecular level. The method is general and not limited to symmetric, or regularly ordered structures. Alone, or in combination with immunoelectron microscopy and electron spectroscopic imaging, electron microscope tomography is a powerful technique in cell and molecular biology.