The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

The herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 µg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.

Single particle analysis of herpes simplex virus: comparing the dimensions of one and the same virions via atomic force and scanning electron microscopy.

Currently, two types of direct methods to characterize and identify single virions are available: electron microscopy (EM) and scanning probe techniques, especially atomic force microscopy (AFM). AFM in particular provides morphologic information even of the ultrastructure of viral specimens without the need to cultivate the virus and to invasively alter the sample prior to the measurements. Thus, AFM can play a critical role as a frontline method in diagnostic virology. Interestingly, varying morphological parameters for virions of the same type can be found in the literature, depending on whether AFM or EM was employed and according to the respective experimental conditions during the AFM measurements. Here, an inter-methodological proof of principle is presented, in which the same single virions of herpes simplex virus 1 were probed by AFM previously and after they were measured by scanning electron microscopy (SEM). Sophisticated chemometric analyses then allowed a calculation of morphological parameters of the ensemble of single virions and a comparison thereof. A distinct decrease in the virions' dimensions was found during as well as after the SEM analyses and could be attributed to the sample preparation for the SEM measurements. Graphical abstract The herpes simplex virus is investigated with scanning electron and atomic force microscopy in view of varying dimensions.

Subassemblies and asymmetry in assembly of herpes simplex virus procapsid.

UNLABELLED: The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2ß heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes' departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation. IMPORTANCE: This paper addresses the molecular mechanisms that govern the self-assembly of large, structurally complex, macromolecular particles, such as the capsids of double-stranded DNA viruses. Although they may consist of thousands of protein subunits of many different kinds, their assembly is precise, ranking them among the largest entities in the biosphere whose structures are uniquely defined to the atomic level. Assembly proceeds in two stages: formation of a precursor particle (procapsid) and maturation, during which major changes in structure and composition take place. Our analysis of the HSV procapsid by cryo-electron microscopy suggests a hierarchical pathway in which multisubunit "protomers" are the building blocks of the procapsid but their subunits are redistributed into different subcomplexes upon being incorporated into a nascent procapsid and are redistributed again in maturation. Assembly is a highly virus-specific process, making it a potential target for antiviral intervention.

Structural analysis of herpes simplex virus by optical super-resolution imaging.

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

Characterization of an oncolytic herpes simplex virus drug candidate.

The structural integrity and conformational stability of a genetically modified live, oncolytic herpes simplex virus (o-HSV) were investigated across a wide pH (5.5-8.0) and temperature (10°C-87.5°C) range. A combination of circular dichroism, intrinsic and extrinsic fluorescence, and static light scattering results was visualized using an empirical phase diagram approach to provide a global assessment of physical stability. Distinct phases were identified including the native state of the virus, an intermediate phase that could represent gradual swelling and/or shedding of the viral envelope, and a highly disrupted, aggregated phase. The nature of these altered forms of the virus was further evaluated by transmission electron microscopy and viral plaque assays. The effect of freeze-thaw (F/T) stress on o-HSV was also examined. After one F/T cycle, a loss of infectious virus titers was observed. In addition, the monomeric virus particle concentration decreased during F/T stress, whereas there was a concurrent increase in larger particles (2-10 µm). The comprehensive biophysical characterization of viral stability conducted in this study identified major degradation events leading to loss of infectivity of o-HSV and represents an important step toward stabilization of the virus against thermal and F/T stresses.

Herpes simplex virus capsid assembly and DNA packaging: a present and future antiviral drug target.

Herpes simplex virus (HSV) is an important pathogenic agent that causes recurrent oral and genital lesions, blindness and encephalitis. It is a member of the family Herpesviridae, which contains three subfamilies (alpha- beta- and gammaherpesvirinae) whose members infect humans to cause a variety of ailments, from benign rashes to nasopharyngeal carcinoma. Although this review focuses on HSV, the assembly steps that occur in the nucleus and the proteins involved are highly conserved among all family members, which suggests that antiviral agents that block these steps might be effective against many different herpesviruses and their associated diseases. Despite this potential, a broadly effective compound has yet to be realized, in part because many of the processes are only poorly understood in sufficient molecular detail. The goal of this review is to outline these intranuclear assembly steps and illustrate potential and existing antiviral strategies that exploit them.

Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.

Nanoparticles reveal that human cervicovaginal mucus is riddled with pores larger than viruses.

The mechanisms by which mucus helps prevent viruses from infecting mucosal surfaces are not well understood. We engineered non-mucoadhesive nanoparticles of various sizes and used them as probes to determine the spacing between mucin fibers (pore sizes) in fresh undiluted human cervicovaginal mucus (CVM) obtained from volunteers with healthy vaginal microflora. We found that most pores in CVM have diameters significantly larger than human viruses (average pore size 340 +/- 70 nm; range approximately 50-1800 nm). This mesh structure is substantially more open than the 15-100-nm spacing expected assuming mucus consists primarily of a random array of individual mucin fibers. Addition of a nonionic detergent to CVM caused the average pore size to decrease to 130 +/- 50 nm. This suggests hydrophobic interactions between lipid-coated "naked" protein regions on mucins normally cause mucin fibers to self-condense and/or bundle with other fibers, creating mucin "cables" at least three times thicker than individual mucin fibers. Although the native mesh structure is not tight enough to trap most viruses, we found that herpes simplex virus (approximately 180 nm) was strongly trapped in CVM, moving at least 8,000-fold slower than non-mucoadhesive 200-nm nanoparticles. This work provides an accurate measurement of the pore structure of fresh, hydrated ex vivo CVM and demonstrates that mucoadhesion, rather than steric obstruction, may be a critical protective mechanism against a major sexually transmitted virus and perhaps other viruses.

Virion disruption by ozone-mediated reactive oxygen species.

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Uncoating the herpes simplex virus genome.

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.

Formation and reversible dissociation of coiled coil of peptide to the C-terminus of the HSV B5 protein: a time-resolved spectroscopic analysis.

An understanding of the molecular mechanisms of the newly characterized herpes simplex virus (HSV) B5 protein is important to further elucidate the HSV cell entry and infection. The synthetic peptide of B5 (wtB5) was functionalized with the nonlinear optical chromophore cascade yellow and its molecular dynamics was probed at physiological and endosomal pH (pH 7.4 and 5.5, respectively). Steady-state CD spectroscopy was utilized to characterize the peptides at different pH. These spectra showed structural changes in the peptide with time measured over several days. Nonlinear optical measurements were carried out to probe the interactions and local environment of the labeled peptide, and the increase in the two-photon cross section of this system suggests an increase in chromophore-peptide interactions. Time-resolved fluorescence upconversion measurements reflected changes in the hydrophilic and hydrophobic local environments of the labeled peptide-chromophore system. Ultrafast depolarization measurements gave rotational correlation times indicative of a reversible change in the size of the peptide. The time-resolved results provide compelling evidence of a reversible dissociation of the coiled coils of the wtB5 peptide. This process was found to be pH-insensitive. The data from this unique combination of techniques provide an initial step to understanding the molecular dynamics of B5 and a framework for the development of novel imaging methods based on two-photon emission, as well as new therapeutics for HSV.

Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro.

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.

A resolution criterion for electron tomography based on cross-validation.

Despite much progress in electron tomography, quantitative assessment of resolution has remained a problematic issue. The criteria that are used in single particle analysis, based on gauging the consistency between density maps calculated from half data sets, are not directly applicable because of the uniqueness of a tomographic volume. Here, we propose two criteria based on a cross-validation approach. One, called FSC(e/o), is based on a Fourier shell correlation comparison between tomograms calculated from the even and odd members of a tilt series. The other, called noise-compensated leave-one-out (NLOO), is based on Fourier ring correlation comparisons between an original projection and the corresponding reprojection of the tomogram calculated from all the other projections, taking into account the differing noise statistics. Plotted as a function of tilt angle, they allow assessment of the angular dependence of resolution and quality control over the series of projections. Integrated over all projections, the results give a global figure for resolution. Tests on simulated tomograms established consistency between these criteria and the FSC(ref), a correlation coefficient calculated between a known reference structure and the corresponding portion of a tomogram containing that structure. The two criteria-FSC(e/o) and NLOO-are mutually consistent when residual noise is the major resolution-limiting factor. When the size of the tilt increment becomes a significant factor, NLOO provides a more reliable criterion, as expected, although it is computationally intensive. Applicable to entire tomograms or selected structures, NLOO has also been tested on experimental tomographic data.

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy.

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.

The antiviral agent 5-chloro-1,3-dihydroxyacridone interferes with assembly and maturation of herpes simplex virus.

Antiviral drug screening and exploratory mechanistic work identified 5-chloro-1,3-dihydroxyacridone as a lead inhibitor of herpes simplex virus (HSV) replication, one without a primary effect on either HSV DNA or late viral protein synthesis (Antivir. Res. 45 (2000) 123). In this report, drug effects on viral DNA cleavage and packaging, HSV capsid production and virion morphogenesis in infected Vero cells were studied systematically in order to better localize the sensitive stage of the replication cycle. Maturation of replicating HSV DNA and virion production at late times were inhibited in the same dose-dependent fashion, suggesting that the drug might directly inhibit the cleavage and packaging processes. Based on density centrifugation analysis however, this possibility appears unlikely because overproduction of neither A- or B-capsids occurred upon drug treatment. Interestingly, similar studies coupled with either Western immunoblot or ultrastructural analysis showed that B-capsids with apparent normal protein composition accumulated at reduced levels (maximally about two- to three-fold) in drug-treated cells. Limited attempts to isolate drug-resistant viral mutants using standard approaches proved unsuccessful. In summery, 5-chloro-1,3-dihydroxyacridone inhibits one or more steps of HSV assembly since treatment results in reduced levels of capsids (particularly B-type) and reduced levels of encapsidated DNA. The action of the acridone derivative is an unusual one, with distinctive features when compared to a recently reported class of HSV encapsidation inhibitor and to the late replication defects of relevant viral mutants.

Targeting gene expression using HSV vectors.

Herpes simplex virus (HSV) is an encapsulated DNA virus, with many favourable properties for use as a gene transfer vector. For gene therapy applications, it may be desirable to restrict transgene expression to pre-defined subsets of cells. One potential method for achieving targeted transgene expression using the HSV vector system might involve dictating the cell types to which the vector will transfer the therapeutic transgene of interest. HSV delivers its genetic payload to cells directly through the plasmalemma; the mechanisms are complex and involve multiple viral and cell surface determinants. We have investigated several ways in which each component of the cell entry cascade may be manipulated in order to restrict viral DNA and transgene delivery to particular cellular populations. Our results indicate that targeted transduction may be a viable approach to achieving our goal of targeted HSV-mediated transgene expression.