Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis

Electron microscopy(EM) should be used in the front line for detection of agents in emergencies and bioterrorism,on accounts of its speed and accuracy.However,the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results.Therefore,the research on viral structure and morphology has become important in EM diagnostic practice.EM has several technological advantages,and should be a fundamental tool in clinical diagnosis of viruses,particularly when agents are unknown or unsuspected.In this article,we review the historical contribution of EM to virology,and its use in virus differentiation,localization of specific virus antigens,virus-cell interaction,and viral morphogenesis.It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy.Furthermore,avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.     

Entry and Disassembly of Large DNA Viruses: Electron Microscopy Leads the Way

Nucleocytoplasmic large DNA viruses are a steadily growing group of viruses that infect a wide range of hosts and are characterized by large particle dimensions and genome sizes. Understanding how they enter into the host cell and deliver their genome in the cytoplasm is therefore particularly intriguing. Here, we review the current knowledge on the entry of two of the best-characterized nucleocytoplasmic large DNA viruses: the poxvirus Vaccinia virus (VACV) and the giant virus Mimivirus. While previous studies on VACV had proposed both direct fusion at the plasma membrane and endocytosis as entry routes, more recent biochemical and morphological data argue for macropinocytosis as well. Notably, direct imaging by electron microscopy (EM) also supported the existence of parallel ways of entry for VACV. Instead, all the giant viruses studied so far only enter cells by phagocytosis as observed by EM, and we discuss the mechanisms for opening of the particle, fusion of the viral and phagosomal membranes and genome delivery via a unique portal, specific for each giant virus. VACV core uncoating, in contrast, remains a morphologically ill-defined process. We argue that correlated light and electron microscopy methods are required to study VACV entry and uncoating in a direct and systematic manner. Such EM studies should also address whether entry of single particles and viral aggregates is different and thus provide an explanation for the different modes of entry described in the literature.     

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.     

Negative staining in the detection of viruses in clinical specimens

Viruses have unique morphology and are therefore good candidates for negative staining. Negative staining with phosphotungstic acid (PTA) or uranyl acetate has facilitated the detection of many viruses in clinical specimens. Enhancement procedures have included the use of centrifugation and agar diffusion for concentrating virus particles, the use of solid phase capture reagents to trap virus particles and the use of secondary antibodies and electron dense markers to help visualize them. Techniques currently in use and employing negative staining include direct EM, immune electron microscopy (IEM), solid phase immune electron microscopy (SPIEM), colloidal gold-labeled protein A (PAG), solid phase IEM employing a second decorator antibody (SPIEMDAT), and solid phase IEM using colloided gold-labeled secondary antibodies (SPEIMDAGT). IEM methods assist with the detection of small viruses or viruses present in low numbers while PAG offers increased sensitivity over direct EM and IEM. In our experience the serum-in-agar (SIA) method is the most sensitive of the PAG IEM techniques for detection of rotavirus particles in clinical specimens. SPIEMDAT enhances the detection of small viruses which are often missed by other techniques due to background staining in specimens. SPEIMDAGT employing colloidal gold-labeled secondary antibody has increased sensitivity and offers the advantage of detecting viral antigen when whole virus particles are not visible. IEM techniques have recently been used for typing viruses using either monospecific antisera or monoclonal antibodies and colloidal gold-labeled secondary antibody.     

Host cell factors and functions involved in vesicular stomatitis virus entry

Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although associated with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-associated proteins and cellular functions.     

Cellular autophagy machinery is not required for vaccinia virus replication and maturation

The origin of the primary membrane of the vaccinia virus, a double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane originates from the autophagic membrane. Morphologic studies by electron microscopy (EM) showed no apparent difference in viral maturation in the autophagy-deficient cell lines, the atg5(-/-) mouse embryonic fibroblasts (MEFs) and the beclin1(-/-) embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, viral growth curves demonstrated that vaccinia viruses replicate and mature in the autophagy-deficient cell lines as efficiently as they do in their isogenic wild type counterpart cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane does not originate from the autophagic membrane.     

The envelope of intracellular African swine fever virus is composed of a single lipid bilayer

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments

The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.     

利用原子力显微镜对A型流感病毒的形态学研究

利用透射电子显微镜(TEM)和原子力显微镜(AFM)观察流感病毒(H1N1),探讨AFM在病毒形态研究中的应用,为病毒形态学研究提供一种新型、简便、快捷的工具.TEM采用磷钨酸负染方法,AFM采用轻敲模式在大气常温下扫描成像,并对主要指标长度(直径)、Ra、Rq等进行测量.两种方法最终得到相似的形态学结果,流感病毒呈球状、丝状,并有一些形状介于两者之间.TEM提供了流感病毒二维图像,可见钉状突起,AFM则呈现了流感病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.与TEM观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具,其表征参数可以作为病毒形态学研究的量化指标.     

Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water

The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3' end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.     

Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.     

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapox- and orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera.     

Structural basis for HIV‐1 DNA integration in the human genome,role of the LEDGF/P75 cofactor

Integration of the human immunodeficiency virus (HIV‐1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild‐type full‐length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA‐binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral‐induced pathologies.     

Wrapping things up about virus RNA replication

All single-stranded 'positive-sense' RNA viruses that infect mammalian, insect or plant cells rearrange internal cellular membranes to provide an environment facilitating virus replication. A striking feature of these unique membrane structures is the induction of 70-100 nm vesicles (either free within the cytoplasm, associated with other induced vesicles or bound within a surrounding membrane) harbouring the viral replication complex (RC). Although similar in appearance, the cellular composition of these vesicles appears to vary for different viruses, implying different organelle origins for the intracellular sites of viral RNA replication. Genetic analysis has revealed that induction of these membrane structures can be attributed to a particular viral gene product, usually a non-structural protein. This review will highlight our current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA-membrane interactions observed between the virus families Flaviviridae and Picornaviridae.     

Epstein-Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes

Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.     

Electron tomography of viruses

Understanding the molecular architectures of enveloped and complex viruses is a challenging frontier in structural biology. In these viruses, the structural and compositional variation from one viral particle to another generally precludes the use of either crystallization or image averaging procedures that have been successfully implemented in the past for highly symmetric viruses. While advances in cryo electron tomography of unstained specimens provide new opportunities for identification and molecular averaging of individual subcomponents such as the surface glycoprotein spikes on purified viruses, electron tomography of stained and plunge-frozen cells is being used to visualize the cellular context of viral entry and replication. Here, we review recent developments in both areas as they relate to our understanding of the biology of heterogeneous and pleiomorphic viruses.