Biomedical subjects

L W Enquist

Publications and source records attributed to L W Enquist.

At least 19 recordsLinked to original sources

PubMed2006

Pseudorabies virus EP0 protein counteracts an interferon-induced antiviral state in a species-specific manner.

Pseudorabies virus (PRV), an alphaherpesvirus related to herpes simplex virus type 1 and varicella-zoster virus, infects a broad host range of mammals. A striking characteristic of PRV infection is the different symptoms and outcomes of infection in natural and nonnatural hosts. Adult pigs, the natural hosts of PRV, survive infection with only mild respiratory symptoms, while nonnatural hosts, including rodents and cattle, invariably die after exhibiting neurological symptoms. Here, we show that the PRV EP0 protein is necessary to overcome an interferon-mediated antiviral response in primary cells from the natural host of PRV but is not necessary in nonnatural-host cells.

Animals↗

PubMed2006

Role of pseudorabies virus Us3 protein kinase during neuronal infection.

The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

Animals↗

PubMed2006

Suppression of the interferon-mediated innate immune response by pseudorabies virus.

Pseudorabies virus (PRV) is an alphaherpesvirus related to the human pathogens herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. PRV is capable of infecting and killing a wide variety of mammals. How it avoids innate immune defenses in so many hosts is not understood. While the anti-interferon (IFN) strategies of HSV-1 have been studied, little is known about how PRV evades the IFN-mediated immune response. In this study, we determined if wild-type PRV infection can overcome the establishment of a beta interferon (IFN-beta)-induced antiviral state in primary rat fibroblasts. Using microarray technology, we found that the expression of a subset of genes normally induced by IFN-beta in these cells was not induced when the cells were simultaneously infected with a wild-type PRV strain. Expression of transcripts associated with major histocompatibility complex class I antigen presentation and NK cell activation was reduced, while transcripts associated with inflammation either were unaffected or were induced by viral infection. This suppression of IFN-stimulated gene expression occurred because IFN signal transduction, in particular the phosphorylation of STAT1, became less effective in PRV-infected cells. At least one virion-associated protein is involved in inhibition of STAT1 tyrosine phosphorylation. This ability to disarm the IFN-beta response offers an explanation for the uniform lethality of virulent PRV infection of nonnatural hosts.

Animals↗

PubMed2005

An in vitro system to study trans-neuronal spread of pseudorabies virus infection.

The neuronal spread of infection of alpha herpesviruses is controlled by unknown mechanisms. In the natural host, primary infection always leads to invasion of the peripheral nervous system, but rarely results in extensive invasion of the central nervous system. After reactivation of latent infection in the peripheral nervous system, virions are produced and shed from epithelial surfaces, but rarely invade the central nervous system. We have been studying two aspects of the general problem. First, using GFP and mRFP fusion proteins, we have used video confocal microscopy to assess mechanisms that influence spread of pseudorabies (PRV) virion components within axons. Second, and the subject of this report, is the development of a new in vitro cell culture system that enables the study of trans-neuronal spread of infection from neurons to non-neuronal cells similar to what happens after reactivation and spread to epithelial surfaces. We have developed a tissue culture system involving tri-chamber Teflon rings that enables facile analysis of trans-neuronal spread. The system duplicates all the known in vivo correlates of trans-neuronal spread and provides the opportunity to do both quantitative and qualitative assessment of spread of PRV infection from infected neurons to non-neuronal cells.

Animals↗

PubMed2005

Olfactory inputs to hypothalamic neurons controlling reproduction and fertility.

In order to gain insight into sensory processing modulating reproductive behavioral and endocrine changes, we have aimed at identifying afferent pathways to neurons synthesizing luteinizing hormone-releasing hormone (LHRH, also known as gonadotropin-releasing hormone [GnRH]), a key neurohormone of reproduction. Injection of conditional pseudorabies virus into the brain of an LHRH::CRE mouse line led to the identification of neuronal networks connected to LHRH neurons. Remarkably, and in contrast to established notions on the nature of LHRH neuronal inputs, our data identify major olfactory projection pathways originating from a discrete population of olfactory sensory neurons but fail to document any synaptic connectivity with the vomeronasal system. Accordingly, chemosensory modulation of LHRH neuronal activity and mating behavior are dramatically impaired in absence of olfactory function, while they appear unaffected in mouse mutants lacking vomeronasal signaling. Further visualization of afferents to LHRH neurons across the brain offers a unique opportunity to uncover complex polysynaptic circuits modulating reproduction and fertility.

Animals↗

PubMed2005

Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22.

Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.

Actins↗

PubMed2005

Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E.

Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.

Animals↗

PubMed2005

Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system.

Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.

Animals↗

PubMed2005

Heterogeneity of a fluorescent tegument component in single pseudorabies virus virions and enveloped axonal assemblies.

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

Animals↗

PubMed2004

Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons.

The core structures of many viruses move within cells by association with host cytoskeletal motor proteins; however, the mechanisms by which intracellular viral particles are transported toward sites of replication or the cell periphery at distinct stages of infection remain to be understood. The regulation of herpesvirus directional transport in sensory neurons was examined by tracking individual viral capsids within axons at multiple frames per s. After entry into axons, capsids underwent bidirectional and saltatory movement to the cell body independently of endosomes. A comparison of entry transport to a previous analysis of capsid axonal transport during egress revealed that capsid targeting in and out of cells occurs by modulation of plus-end, but not minus-end, motion. Entry transport was unperturbed by the presence of egressing virus from a prior infection, indicating that transport direction is not modulated globally by viral gene expression, but rather directly by a component of the viral particle.

Animals↗

PubMed2004

Two modes of pseudorabies virus neuroinvasion and lethality in mice.

We describe two distinct modes of neuroinvasion and lethality after murine flank inoculation with virulent and attenuated strains of pseudorabies virus (PRV). Mice infected with virulent (e.g., PRV-Becker, PRV-Kaplan, or PRV-NIA3) strains self-mutilate their flank skin in response to virally induced pruritus, die rapidly with no identifiable symptoms of central nervous system (CNS) infection such as behavioral abnormalities, and have little infectious virus or viral antigen in the brain. In distinct contrast, animals infected with an attenuated PRV vaccine strain (PRV-Bartha) survive approximately three times longer than wild-type PRV-infected animals, exhibit severe CNS abnormalities, and have an abundance of infectious virus in the brain at the time of death. Interestingly, these animals have no skin lesions and do not appear pruritic at any time during infection. The severe pruritus and relatively earlier time until death induced by wild-type PRV infection may reflect the peripheral nervous system (PNS) and immune responses to infection rather than a fatal, virally induced CNS pathology. Based on previously characterized afferent (sensory) and efferent (motor) neuronal pathways that innervate the skin, we deduced that wild-type virulent strains transit through the PNS via both afferent and efferent routes, whereas PRV-Bartha travels by only efferent routes in the PNS en route to the brain.

Animals↗

PubMed2004

Cyclooxygenase-1 and -2 are required for production of infectious pseudorabies virus.

We have recently shown that cyclooxygenase-2 (COX-2) transcription is markedly induced after herpes simplex virus type 1 and pseudorabies virus (PRV) infections of rat embryonic fibroblast (REF) cells (N. Ray and L. W. Enquist, J. Virol. 78:3489-3501, 2004). For this study, we investigated the role of cyclooxygenase induction in the replication and growth of PRV. We demonstrate here a concordant increase in COX-2 mRNA and protein levels after the infection of REF cells. Inhibitors blocking the activity of cyclooxygenases caused a dramatic reduction in PRV growth. Viral growth could be restored if prostaglandin E(2), the final product of COX-2 activity, was added simultaneously with the COX inhibitors. Immediate-early protein IE180, major capsid protein VP5, and glycoprotein expression were slightly reduced in the presence of COX-2 inhibitors, but expression of the early protein EP0 was not affected by COX inhibition. Viral DNA replication was marginally reduced in the presence of a COX-1/2 inhibitor, but there was no defect in viral DNA cleavage. Electron microscopy analysis revealed an increased number of unusual empty capsid structures in the nuclei of cells infected with PRV in the presence of a COX-1/2 inhibitor. These capsid structures shared some characteristics with procapsids but had a novel appearance by negative staining. Our data establish a role for COX-1 and COX-2 in facilitating the efficient growth and replication of PRV in primary cells.

Animals↗

PubMed2004

Transcriptional response of a common permissive cell type to infection by two diverse alphaherpesviruses.

Pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) are distantly related alphaherpesviruses whose natural hosts are pigs and humans, respectively. Adult infections of natural hosts are mild and rarely lethal. However, both viruses are also able to infect other hosts, often with lethal effects. In this report, we use the paradigm of infection of a common permissive cell type and microarray analysis to determine if these two diverse alphaherpesviruses engage similar or different cellular pathways to obtain a common outcome: productive infection. We compared cellular gene expression in growth-arrested, primary rat embryonic fibroblasts that were mock infected or infected with either purified PRV-Becker or HSV-1(F). Infections by either virus affect the transcription of more than 1,500 cellular genes by threefold or more. Few differences are detected early, and the majority of changes occur during the late stages of infection. Remarkably, the transcripts of about 500 genes are regulated in common, while the rest are regulated in a virus-specific manner. Genes whose expression is affected by infection fall into a diverse group of functional classes and cellular pathways. Furthermore, a comparison of the cellular response to HSV-1 infection of primary human and rat fibroblasts revealed unexpected diversity in the transcript profiles.

Animals↗

PubMed2003

The pseudorabies virus Us2 protein, a virion tegument component, is prenylated in infected cells.

The Us2 gene is conserved among alphaherpesviruses, but its function is not known. We demonstrate here that the pseudorabies virus (PRV) Us2 protein is synthesized early after infection and localizes to cytoplasmic vesicles and to the plasma membrane, despite the lack of a recognizable signal sequence or membrane-spanning domain. Us2 protein is also packaged as part of the tegument of mature virions. The Us2 carboxy-terminal four amino acids comprise a CAAX motif, a well-characterized signal for protein prenylation. Treatment of infected cells with lovastatin, a drug that disrupts protein prenylation, changed the relative electrophoretic mobility of Us2 in sodium dodecyl sulfate-polyacrylamide gels. In addition, lovastatin treatment caused a dramatic relocalization of Us2 to cytoplasmic punctate structures associated with microtubules, which appeared to concentrate over the microtubule organizing center. When the CAAX motif was changed to GAAX and the mutant protein was synthesized from an expression plasmid, it concentrated in punctate cytoplasmic structures reminiscent of Us2 localization in infected cells treated with lovastatin. We suggest that prenylation of PRV Us2 protein is required for proper membrane association. Curiously, the Us2 protein isolated from purified virions does not appear to be prenylated. This is the first report to describe the prenylation of an alphaherpesvirus protein.

Amino Acid Motifs↗

PubMed2002

Exploiting circuit-specific spread of pseudorabies virus in the central nervous system: insights to pathogenesis and circuit tracers.

The neurotropic alpha-herpesviruses are common mammalian pathogens that invade the peripheral and central nervous system of their hosts. Their ability to invade and spread in the nervous system in a directional manner has been exploited to develop them as neuronal circuit tracers. Tracing viruses spread among synaptically connected neurons and, by assaying brain sections for viral antigen or reporter genes expressed from the viruses, chains of synaptically connected neurons can be visualized. Virulent field strains generally are not good tracers, but some attenuated strains perform well. Live attenuated vaccine strains of pseudorabies virus (PRV), such as PRV Bartha, are among the most popular virus circuit tracers. It may be counterintuitive that attenuation results in improved neural tracing that requires extensive replication and spread in the brain. This report summarizes two lines of experiments directed to resolving this apparent paradox and introduces a new paradigm for tracing viruses.

Animals↗

PubMed2002

Directional spread of an alpha-herpesvirus in the nervous system.

Pseudorabies virus (PRV), an alpha-herpesvirus, is capable of spreading between synaptically connected neurons in diverse hosts. In this report, two lines of experimentation are summarized that provide insight into the mechanism of virus spread in neurons. First, techniques were developed to measure the transport dynamics of capsids in infected neurons. Individual viral capsids labeled with green fluorescent protein (GFP) were visualized and tracked as they moved in axons away from infected neuronal cell bodies in culture during egress. Second, the effects of three viral membrane proteins (gE, gI and Us9) on the localization of envelope, tegument, and capsid proteins in infected, cultured sympathetic neurons were determined. These three proteins are necessary for spread of infection from pre-synaptic neurons to post-synaptic neurons in vivo (anterograde spread). Us9 mutants apparently are defective in anterograde spread in neural circuits because essential viral membrane proteins such as gB are not transported to axon terminals to facilitate spread to the connected neuron. By contrast, gE and gI mutants manifest their phenotype because these proteins most likely function at the axon terminal of the infected neuron to promote spread. These two sets of experiments are consistent with a model for herpesvirus spread in neurons first suggested by Cunningham and colleagues where capsids and envelope proteins, but not whole virions, are transported separately into the axon.

Animals↗

PubMed2002

The pseudorabies virus VP22 homologue (UL49) is dispensable for virus growth in vitro and has no effect on virulence and neuronal spread in rodents.

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.

Animals↗

PubMed2001

A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.

Pseudorabies virus, an alpha-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9-null mutant infections but not gE-null mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

Amino Acid Sequence↗