Bound Virus Research Articles

Giant Plasma Membrane Vesicles as Cellular‐Mimics for Probing SARS‐CoV‐2 Binding at Single Particle Level

AbstractMembrane receptors, attachment factors and various other membrane factors play a critical role in the specific attachment of virus particles to a cell surface. Researchers have been employing a range of model systems starting from receptor‐reconstituted lipid membranes to live cells for quantitative evaluation of virus‐receptor/attachment factor interactions. In this work, we show that giant plasma membrane vesicles (GPMVs) derived from adherent cells are a suitable cell‐mimicking model system to examine the binding of SARS‐CoV‐2 to cellular receptors. We evaluated the binding of cell‐cultured SARS‐CoV‐2 to GPMVs by imaging single virus particles with the wide‐field fluorescence microscopy technique. The GPMVs were derived from three different cell lines, i. e., Vero, HEK293T and A549. We compared the number of bound virus particles per cell, cell‐attached GPMV, and cell‐free GPMV of three different cell lines. We explain the observed trend in the data from the expression level of endogenous ACE2 protein and also, conclude that the protein is functional in GPMVs for virus and antibody binding.

Hepatitis delta virus RNA decline post-inoculation in human NTCP transgenic mice is biphasic.

Chronic infection with hepatitis B and delta viruses (HDV) is the most serious form of viral hepatitis due to more severe manifestations of an accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. We characterized early HDV kinetics post-inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor-human sodium taurocholate co-transporting polypeptide (hNTCP). Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific-binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 35 minutes (standard error, SE: 6.3), binds to non-specific cells with a rate of 0.05 per hour (SE: 0.01), and returns as free virus with a rate of 0.11 per hour (SE: 0.02). Characterizing early HDV-host kinetics elucidates how quickly HDV is either cleared or bound depending on the immunological background and hNTCP presence. IMPORTANCE The persistence phase of HDV infection has been studied in some animal models; however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post-inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics.

Superchiral near fields detect virus structure

Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry (referred to as superchirality) and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum.

Structural transitions in influenza haemagglutinin at membrane fusionpH.

Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope glycoproteins bind the infecting virion to cell-surface receptors and mediate membrane fusion. In the case of influenza, the receptor-binding glycoprotein is the haemagglutinin (HA), and following receptor-mediated uptake of the bound virus by endocytosis1, it is the HA that mediates fusion of the virus envelope with the membrane of the endosome2. Each subunit of the trimeric HA consists of two disulfide-linked polypeptides, HA1 and HA2. The larger, virus-membrane-distal, HA1 mediates receptor binding; the smaller, membrane-proximal, HA2 anchors HA in the envelope and contains the fusion peptide, a region that is directly involved in membrane interaction3. The low pH of endosomes activates fusion by facilitating irreversible conformational changes in the glycoprotein. The structures of the initial HA at neutral pH and the final HA at fusion pH have been investigated by electron microscopy4,5 and X-ray crystallography6–8. Here, to further study the process of fusion, we incubate HA for different times at pH 5.0 and directly image structural changes using single-particle cryo-electron microscopy. We describe three distinct, previously undescribed forms of HA, most notably a 150 Å-long triple-helical coil of HA2, which may bridge between the viral and endosomal membranes. Comparison of these structures reveals concerted conformational rearrangements through which the HA mediates membrane fusion.

Влияния различных факторов на сорбционные взаимодействия электропроводящего полимера с вирусами гриппа

Для сорбции вирусов гриппа из водной среды применен электропроводящий полимер полипиррол с сопряженной структурой цепей. Исследована эффективность связывания вируса полимером для чего, использованы различные факторы, разрушающие сорбционные взаимодействия: воздействие растворителя, повышенная температура, ультразвук. Изучена возможность десорбции вируса под действием перечисленных факторов. Определена инфекционная активность вирусов в обработанных сорбентом водных средах, в комплексах с полимером, у десорбированных вирусов

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

107. A Computational Model of Adenovirus Infection

Computational modeling of biological processes provides two distinct benefits for empirical investigators. First, computational models test the impact of an immense array of variables at a broad range of values rapidly and inexpensively in silico. Second, a computational model teaches scientists about the magnitude of an impact due to changes in values of variables. The goal of this project is to develop a highly flexible, user-friendly, biologically relevant model of DNA virus infection. Adenovirus is used as a comparator for the purpose of establishing states (distinct environments) and for identifying the processes that permit transfer from state to state, the rates of those transitions, and the three-dimensional position of each virus at each time point. States in this model include free extracellular virus, cell-surface bound virus, endosome virus, lysosome virus, cytosolic virus, microtubule-associated virus, microtubule-organizing center-associated virus, nuclear bound virus, and inactivated virus. Rates governing transitions between states include diffusion rates, binding and dissociation rates, and microtubule-associated motility, among others. The model employs a game controller to introduce stochastic variability around the chosen rate ensuring that no two iterations of the model give the same dataset. Outputs from the model include comma-separated-value (.csv) data files that are amenable to spreadsheet analysis, graphs showing the relative population of viruses in each state (hundreds of viruses can be used in each infection), and three-dimensional images of the infected cell. An initial characterization of the model demonstrates the veracity of several individual elements. For example, with a pre-set half time for endocytic internalization of bound virus from the cell surface, one would predict that 87.5% of bound virus at time zero would enter the cell in 3 half times (7.5 minutes). Three iterations of the model predicted internalization of 84.2 +/- 13.9% (mean +/- sd) of bound virus over that time period. Similarly, modeling diffusion of vectors in the cytoplasm is an important element in understanding the contribution of microtubule-associated motility to DNA delivery to the nucleus. A prior study of adenovirus diffusion in aqueous medium indicated a diffusion coefficient of 3.67 +/- 0.03 × 10^-8 cm2sec-1 (Oliver et al., 1976, BBA 437:589). The cytoplasm is known to impede diffusion, especially for particles like adenovirus that are larger than 50 nm (Seksek et al., 1997, JCB 138:131), leading to a reduction of the diffusion coefficient by at least two orders of magnitude. Using conditions that allow for a total linear translocation of 1000 nm per sec with a stochastic re-setting of direction 200 times per sec, three iterations of the model predict a diffusion coefficient of 1.80 +/-0.48 × 10^-11 cm2sec-1 (mean +/- se), 204x less than diffusion of adenovirus in water. The model will be validated against published experimental data and will incorporate point-and-click selection of infection conditions. In summary, the model provides a user-friendly, visual and quantitative tool for performing computational studies of viral infection.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry.

Modular glycosphere assays for high-throughput functional characterization of influenza viruses

BackgroundThe ongoing global efforts to control influenza epidemics and pandemics require high-throughput technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional indicator for the relative hazard imposed by circulating and newly emerging influenza strains.ResultsStreptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-throughput analyses of influenza virus produced dose–response curves that allow for functional assessment of relative receptor affinity and thus transmissibility.ConclusionsModular glycosphere assays for high-throughput functional characterization of influenza viruses introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards to receptor specificity, host adaptation, and virus transmissibility.

Phosphatidylinositol 4-Kinase IIIβ Is Required for Severe Acute Respiratory Syndrome Coronavirus Spike-mediated Cell Entry

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection.

P04-19. Analysis of the binding of multiple clades of HIV-1 by a modified virus capture assay

Methods Protein G plates were coated with 2F5, 4E10, 2G12, 21.2 (pooled positive HIV-1 human sera), or with anti-IgM antibodies. Primary HIV-1 or pseudoviruses (pv) from clades A [92UG(pv), 93RW(pv)], B [MN, US-1, MN(pv), US-1(pv)], C [GS016], and D [57128(pv)], A07412(pv)] were then added to the plates in the presence or absence of sCD4 to determine the ability of the antibodies to capture virus. The amount of bound virus was determined by a p24 ELISA. The appropriate viruses were also tested in a TZM-bl neutralization assay using mAbs 2F5 and 4E10.

Immobilized HIV‐1 Tat protein promotes gene transfer via a transactivation‐independent mechanism which requires binding of Tat to viral particles

Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism. Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay. Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env-CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants. Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy.

What we still don't know about AIDS

A great deal is known about the human immunodeficiency virus (HIV) that causes acquired immune deficiency syndrome (AIDS) [1], one of whose cardinal features is its exquisite adaptation to its human host. It enters the body through damaged epithelia, or more insidiously, through specialized cells (M cells) in the intestinal epithelium whose function is to deliver viruses and bacteria to waiting immune cells in the tissue below. There, the virus binds to a specialized receptor on the surface of one of these cells - the dendritic cells, which play a central part in activating the CD4+ T lymphocytes whose destruction by the virus ultimately and lethally disables the immune system. Recognition of the bound virus causes the dendritic cell to migrate to the lymphoid tissues where it engages with the CD4+ T lymphocytes that it activates. This enables the virus to bind to molecules on the surface of the T cell - a highly specific interaction involving the CD4 molecules that give CD4+ T cells their name and that enables the virus to enter the cell. Once the virus is inside the cell, it produces DNA copies of its genome that integrate into the host DNA where cellular transcriptional regulators specifically induced by activation of CD4+ T cells are instrumental in activating transcription of the viral genome to produce more viruses. A great deal is unknown about what happens to the adaptive immune system in consequence of this fiendish and focused assault. The adaptive immune system consists of the lymphocytes that provide lasting immunity, and CD4+ T lymphocytes are essential to activating most of adaptive immunity. The loss of these cells in HIV infection thus punches an enormous hole in the immune defences of the host. But CD4+ T cells are homeostatically self-renewing, and it is still unclear exactly why they are progressively lost in HIV infection. It is also unclear what happens to the dynamics of the immune system. HIV infection, paradoxically, is characterized by generalized immune activation the basis for which is not understood. These lacunae in understanding are due at least partly to limitations to the research tools available to investigate these phenomena. Because of its intimate and specific dependence on the host cell machinery, HIV infects only humans, although there is a monkey counterpart, SIV, that produces a syndrome similar to AIDS in rhesus macaques. (Rhesus macaques are not the natural host of SIV: in sooty mangabeys, which are, it causes little inconvenience - another phenomenon that is not understood.) So AIDS can be investigated only in humans and monkeys, which are cumbersome and limited experimental tools. Mice, whose immune systems are the best-understood in the animal kingdom and which offer ease of maintenance and manipulation, have no equivalent pathogen. With this in mind, George Kassiotis and colleagues set out to generate a mouse in which, as in human HIV infection, CD4+ T cells are killed on activation. They did this by a feat of genetic engineering involving Cre-lox and the A fragment of diphtheria toxin (DTA) and which is described both in their paper in this issue of Journal of Biology [2] and, more succinctly, in the accompanying commentary [3]. Investigation of the engineered mouse has led them to the conclusion that at least some of the disturbance to immune dynamics in HIV infection may be due to the preferential destruction of a highly topical and much-invoked subset of CD4+ T cells called regulatory T cells. In the accompanying commentary, Nienke Vrisekoop, Judith Mandl and Ronald Germain explain what is known and what is not known about immune dynamics in HIV infection, where the mouse results are and are not consistent with what we know, and what the value of this tool may be. One way or another, the CD4+ T cell conditional mutant mouse may help to close the gap between the detailed understanding of the molecular and cell biology of HIV infection, and the very imperfect understanding of the impact of HIV on T cell dynamics.

Concentrating rotaviruses from water samples using monolithic chromatographic supports

Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml.

Virus immunocapture provides evidence of CD8 lymphocyte-derived HIV-1 in vivo

To demonstrate that HIV-1 immunocapture with an antibody against CD8 specifically captures virions derived from infected CD8 T cells, and to determine the proportion of HIV-1 derived from CD8 lymphocytes in plasma samples from HIV-infected individuals. A virus capture method was developed to enable the detection of HIV-1 virions based upon the presence of certain cell-specific host-derived proteins (CD8, CD3, CD36) within the viral envelope. HIV-1 virions were captured using antibodies against these proteins and levels of bound virus were determined by quantitative reverse transcriptase-polymerase chain reaction. Highly pure CD8 and CD3+CD8- T-cell cultures were used as in-vitro models to determine the specificity of the virus capture technique. The in-vitro model demonstrates that incorporation of the CD8 molecule into released virions is specific to infection of CD8 T cells. Levels of HIV-1 immunocaptured from plasma of infected individuals using the anti-CD8 antibody indicate that up to 15% (range 10-33) of the plasma viral load is derived from CD8 lymphocytes. This study demonstrates for the first time that HIV-1-infected CD8 T cells can contribute substantially to levels of circulating virus during the course of infection. Levels of CD8-derived virus did not correlate with the level of infection of circulating CD8 T cells, but do show a significantly good fit to plasma viral loads based on a power model. The extensive infection of CD8 T cells implied by these results may contribute towards immune dysfunction and disease progression to AIDS.

Matrix Fibronectin Binds Gammaretrovirus and Assists in Entry: New Light on Viral Infections

A major entry route for the gammaretrovirus amphotropic murine leukemia virus (A-MLV) into NIH 3T3 fibroblasts is via caveola-dependent endocytosis. However, during the infection time, few viral particles can be observed intracellularly. Analyzing the dynamics of the A-MLV infection process by using total internal reflection fluorescence microscopy, we show that the majority of viruses are extracellular and bound to the fibronectin matrix. Moreover, the amounts of bound virus and of fibronectin correlated. Using confocal microscopy, nanoparticles targeted to fibronectin by a III1C-fibronectin fragment or anti-fibronectin antibody were detected intracellularly in NIH 3T3 cells; unconjugated nanoparticles neither bound to cells nor were detectable intracellularly. Furthermore, A-MLV colocalized intracellularly with the fibronectin-targeted nanoparticles, suggesting that they were taken up by the same cellular pathway. Both A-MLV entry and fibronectin turnover depend on caveolar endocytosis, and we found that inhibiting viral binding to the extracellular NIH 3T3 fibronectin-matrix dramatically reduced A-MLV infection, indeed, showing an active role of fibronectin in infection. We suggest that binding to the cellular fibronectin matrix provides a new mechanism by which viruses can enter cells.

Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20 mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.

Epstein–Barr virus-induced B-cell transformation: quantitating events from virus binding to cell outgrowth

Epstein-Barr virus (EBV) infection and growth activation of human B cells is central to virus biology and disease pathogenesis, but is poorly understood in quantitative terms. Here, using virus at defined m.o.i., the different stages of this process at the single-cell level are followed in vitro. Virus binding to the B-cell surface, assayed by quantitative PCR, is highly efficient, particularly at the low m.o.i. values that most likely reflect physiologic events in vivo. However, only 10-15 % of bound virus genomes reach the cell nucleus, as visualized by sensitive fluorescence in situ hybridization (FISH) assay; viral genomes acquired per cell nucleus range from 1 to >10, depending on the m.o.i. Thereafter, despite differences in initial genome load, almost all nuclear genome-positive cells then go on to express the virus-encoded nuclear antigen EBNA2, upregulate the cell activation antigen CD23 and transit the cell cycle. EBNA2-positive cells in the first cycle post-infection then grow out to lymphoblastoid cell lines (LCLs) just as efficiently as do cells limiting-diluted from already established LCLs. This study therefore identifies EBV genome delivery to the nucleus as a key rate-limiting step in B-cell transformation, and highlights the remarkable efficiency with which a single virus genome, having reached the nucleus, then drives the transformation programme.

Studying the binding and entry of the intracellular and extracellular enveloped forms of vaccinia virus.

This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)-the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)-using immunofluorescent staining and confocal microscopy. After binding to or penetration of the cells, IMV, EEV, and virus cores are distinguished by different antibodies. Bound virus or penetrated cores are visualized and recorded by confocal microscopy and can be accurately counted. Although specific antibodies to IMV, EEV, and virus cores are required, this method is highly quantitative and also allows the recognition of virus aggregates, which would not be possible using other techniques, such as flow cytometry and radiolabeling of virus particles. Furthermore, this method bypasses the need for EEV purification that may damage the EEV membrane and release an IMV particle.