Single Virus Particle Research Articles

Clinical variability of GM1 gangliosidosis

In the last few decades, flow cytometry has redefined the field of biology, exponentially enhancing our understanding of cells, immunology, and microbiology. Flow cytometry recently gave birth to flow virometry, a new way to detect, analyze, and characterize single viral particles. Detection of viruses by flow cytometry is possible due to improvements in current flow cytometers, calibration, and tuning methods. We summarize the recent birth and novel uses of flow virometry and the progressive evolution of this tool to advance the field of virology. We also discuss the various flow virometry methods used to identify and analyze viruses. We briefly summarize other applications of flow virometry, including: virus detection, quantification, population discrimination, and viral particles’ antigenic properties. Finally, we summarize how viral sorting will allow further progress of flow virometry to relate viral surface characteristics to infectivity properties.

Viral highway to nucleus exposed by image correlation analyses

Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

Redox-Immunofunctionalized Potyvirus Nanoparticles for High-Resolution Imaging by AFM-SECM Correlative Microscopy.

We present in this chapter a new experimental approach allowing the high resolution imaging of immune complexes on virus particles. Combined atomic force-electrochemical microscopy (AFM-SECM) is used to image the presence of ferrocene functionalized specific antibodies on the surface of potyvirus particles. For this purpose, potyviruses, flexuous filamentous phytoviruses with a high aspect ratio, have been chosen. This technique allows analysis of the distribution of antibody labeling over the virus population. But, more importantly, it opens up the imaging of immune complexes decorating a single viral particle. Finally, its high resolution allows the characterization in situ of the ultrastructure of a single immune complex on the particle.

Deciphering Virus Entry with Fluorescently Labeled Viral Particles.

To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.

Flow virometry as a tool to study viruses.

In the last few decades, flow cytometry has redefined the field of biology, exponentially enhancing our understanding of cells, immunology, and microbiology. Flow cytometry recently gave birth to flow virometry, a new way to detect, analyze, and characterize single viral particles. Detection of viruses by flow cytometry is possible due to improvements in current flow cytometers, calibration, and tuning methods. We summarize the recent birth and novel uses of flow virometry and the progressive evolution of this tool to advance the field of virology. We also discuss the various flow virometry methods used to identify and analyze viruses. We briefly summarize other applications of flow virometry, including: virus detection, quantification, population discrimination, and viral particles' antigenic properties. Finally, we summarize how viral sorting will allow further progress of flow virometry to relate viral surface characteristics to infectivity properties.

Membrane Fluidity Sensing on the Single Virus Particle Level with Plasmonic Nanoparticle Transducers.

Viral membranes are nanomaterials whose fluidity depends on their composition, in particular, the cholesterol (chol) content. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of sensing the membrane fluidity on the single-virus level are required. In this manuscript, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature, for the first time on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as a function of time. The rate of chol extraction for individual VLPs showed a broad spread, presumably due to differences in the membrane composition for the individual VLPs, and this heterogeneity increased with decreasing MβCD concentration.

Plating Bacteriophage M13.

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C.

Single-Particle Detection of Transcription following Rotavirus Entry.

ABSTRACTInfectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.

Polarity-Sensitive Probes for Superresolution Stimulated Emission Depletion Microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.

Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2.

ABSTRACTThe hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like β-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a β-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design.

Genomic exploration of individual giant ocean viruses.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

The Envelope Gene of Transmitted HIV-1 Resists a Late Interferon Gamma-Induced Block.

ABSTRACTType I interferon (IFN) signaling engenders an antiviral state that likely plays an important role in constraining HIV-1 transmission and contributes to defining subsequent AIDS pathogenesis. Type II IFN (IFN-γ) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFN-γ stimulation can induce an antiviral state that can be both distinct from that of type I interferon and can potently inhibit HIV-1 in primary CD4+ T cells and a number of human cell lines. Strikingly, we find that transmitted/founder (TF) HIV-1 viruses can resist a late block that is induced by type II IFN, and the use of chimeric IFN-γ-sensitive/resistant viruses indicates that interferon resistance maps to the env gene. Simultaneously, in vitro evolution also revealed that just a single amino acid substitution in the envelope can confer substantial resistance to IFN-mediated inhibition. Thus, the env gene of transmitted HIV-1 confers resistance to a late block that is phenotypically distinct from blocks previously described to be resisted by env and is therefore mediated by unknown IFN-γ-stimulated factor(s) in human CD4+ T cells and cell lines. This important unidentified block could play a key role in constraining HIV-1 transmission.IMPORTANCE The human immune system can hinder invading pathogens through interferon (IFN) signaling. One consequence of this signaling is that cells enter an antiviral state, increasing the levels of hundreds of defenses that can inhibit the replication and spread of viruses. The majority of HIV-1 infections result from a single virus particle (the transmitted/founder) that makes it past these defenses and colonizes the host. Thus, the founder virus is hypothesized to be a relatively interferon-resistant entity. Here, we show that certain HIV-1 envelope genes have the unanticipated ability to resist specific human defenses mediated by different types of interferons. Strikingly, the envelope gene from a founder HIV-1 virus is far better at evading these defenses than the corresponding gene from a common HIV-1 lab strain. Thus, these defenses could play a role in constraining the transmission of HIV-1 and may select for transmitted viruses that are resistant to this IFN-mediated inhibition.

The Influence of Membrane Composition on the Kinetics of Influenza Virus Fusion Measured using a Single Particle Approach

In order to infect a host cell the influenza virus fuses its envelope with the host cell membrane. This fusion of the viral and cell membranes is mediated by the viral surface protein hemagglutinin (HA) which both docks the virus and fuses the membranes. Using Total Internal Reflection Fluorescence (TIRF) microscopy and a planar, fluid bilayer it is possible to study the kinetics of hemifusion in single virus particles. We have used this technique to study the yield and kinetics of the fusion of influenza with multiple different bilayer compositions. This allows us to better understand the physics behind HA mediated membrane fusion, and thus to better understand the process of influenza infection.

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells.

Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm2/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Towards a correlative approach for characterising single virus particles by transmission electron microscopy and nanoscale Raman spectroscopy.

The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a "class I" fusogen) and West Nile virus envelope protein ("class II"). Our study of VSV now extends this description to "class III" viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers

Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach. Fluorescence microscopy and PCR were used to verify expression of HIV-Spinach RNAs in human cells. HIV-1 gag RNA production and fluorescence were measured by qPCR and fluorometry, respectively. HIV-Spinach RNAs were fluorometrically detectable in vitro and were transcribed in human cell lines and primary cells, with both spliced and unspliced species detected by PCR. HIV-Spinach RNAs were visible by fluorescence microscopy in living cells, although signal was reproducibly weak. Cells expressing HIV-Spinach RNAs were capable of producing fluorometrically detectable virions, although detection of single viral particles was not possible. In summary, we have investigated a novel method for detecting HIV RNAs in living cells using the Spinach RNA aptamer. Despite the limitations of the present aptamer/fluorophore combination, this is the first application of this technology to an infectious disease and provides a foundation for future research into improved methods for studying HIV expression.

Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating.

Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.

The trickle before the torrent-diffraction data from X-ray lasers.

Today Scientific Data launched a collection of publications describing data from X-ray free-electron lasers under the theme ‘Structural Biology Applications of X-ray Lasers’. The papers cover data on nanocrystals, single virus particles, isolated cell organelles, and living cells. All data are deposited with the Coherent X-ray Imaging Data Bank (CXIDB) and available to the scientific community to develop ideas, tools and procedures to meet challenges with the expected torrents of data from new X-ray lasers, capable of producing billion exposures per day.