Search PubMed⌕ Search

Biomedical subjects

L W Enquist

Publications and source records attributed to L W Enquist.

At least 73 records · Page 4Linked to original sources
PubMed1995

Dendritic morphology of cardiac related medullary neurons defined by circuit-specific infection by a recombinant pseudorabies virus expressing beta-galactosidase.

The transneuronal herpesvirus tracer, pseudorabies virus (PRV) was used to determine the dendritic architecture of cardiac-related neurons. We constructed a derivative of the Bartha strain of PRV called PRV-BaBlu, that carries the lacZ gene of E. coli. Expression of beta-galactosidase by this recombinant virus enabled us to define the dendritic morphology of motoneurons and interneurons that innervate the heart. beta-galactosidase antigen filled dendritic processes that were clearly revealed by antibodies to beta-galactosidase. In contrast, the standard enzymatic reaction for detection of beta-galactosidase activity stained the cell soma well, but was inferior for labeling dendrites. Following PRV-BaBlu cardiac injection, infected neurons were clearly defined and labeled dendrites could be traced for long distances, sometimes greater than 800 microns from the cell body. Labeled dendrites of cardiomotor neurons primarily located in the nucleus ambiguus (NA) were extensive and sometimes intertwined with dendrites from other labeled motoneurons. Dendrites of labeled neurons in the dorsal motor nucleus of the vagus (DMV) typically extended in the mediolateral direction in the transverse plane. Transynaptically labeled interneurons interposed between the cardiorespiratory region of the nucleus tractus solitarius (NTS) and the NA were primarily located in the NA region and the reticular arc, the area between the DMV and NA. These interneurons had long dendrites extending along the reticular arc in the transverse plane. The dendritic arborizations of infected cardiac-related neurons in the NTS were variable in extent. We conclude that antibody detection of beta-galactosidase expressed by PRV-BaBlu after infection of neural cardiac circuits provides a superior method to define the dendrites and dendritic fields of cardiac-related motoneurons and interneurons.

Animals↗
PubMed1995

Central neuronal circuit innervating the rat heart defined by transneuronal transport of pseudorabies virus.

We defined the central circuit innervating various regions of the rat heart using a neurotropic herpesvirus as a transneuronal tracer. Location of viral antigens in the brain after cardiac injection of three strains of pseudorabies virus (PRV) provided insight into vagal preganglionic neurons and their connected interneurons. At short survival times, labeled vagal preganglionic neurons were localized in both the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus (DMV), and in an arc-like band through the reticular formation between the NA and the DMV. The amount of DMV labeling was dependent on viral strain. Similar distributions of labeled neurons were observed following either ganglionic, sinoatrial node, or ventricular injections. At intermediate survival times postcardiac injection, the virus replicated in vagal preganglionic neurons and was trans-synaptically transported to interneurons observed primarily in the NA regions and in an arc-like band through the reticular formation. Labeled neurons were also observed in ventral regions of the nucleus of the solitary tract (NTS). At longer survival times, labeled neurons were found in various regions of the NTS with the most abundant label dorsal and dorsomedial to the solitary tract. Abundant neuronal labeling was also found in the intermediolateral cell column, the raphe nuclei, the caudal and rostral ventral lateral medulla, the A5 region, the locus coeruleus, and the lateral and paraventricular hypothalamic nuclei. These data define the central circuits including the interneuronal connections that innervate various cardiac targets.

Animals↗
PubMed1995

Neurovirulence of pseudorabies virus.

Virulence is defined as the relative capacity of a microorganism to overcome the defense mechanisms of the host organism and thereby cause disease. Virally induced virulence is usually quantitated by measuring the mean time to death or appearance of symptoms following viral inoculation. In this review we make a distinction between general virulence and neurovirulence. We define neurovirulence as the degree of pathogenesis in the nervous system, but intend it to be more encompassing than the simple ability of the virus to grow in the central nervous system (CNS). This distinction is made possible by recent advances that permit an integrated assessment of the degree of pathology, reactive gliosis, and inflammatory response to infection in the intact organism with specific antisera and molecular probes.

Animals↗
PubMed1994

Innervation of the heart and its central medullary origin defined by viral tracing.

The vagus nerve exerts a profound influence on the heart, regulating the heart rate and rhythm. An extensive vagal innervation of the cardiac ventricles and the central origin and extent of this innervation was demonstrated by transynaptic transport of pseudorabies virus with a virulent and two attenuated pseudorabies viral strains. The neurons that innervate the ventricles are numerous, and their distribution within the nucleus ambiguus and dorsal motor nucleus of the vagus is similar to that of neurons innervating other cardiac targets, such as the sino-atrial node. These data provide a neuroanatomical correlate to the physiological influence of the vagus nerve on ventricular function.

Animals↗
PubMed1994

Complementation analysis of pseudorabies virus gE and gI mutants in retinal ganglion cell neurotropism.

Pseudorabies virus glycoproteins gE and gI are required to infect some, but not all, regions of the rodent central nervous system after peripheral injection. After infection of the retina, pseudorabies virus mutants lacking either gE or gI can subsequently infect neural centers involved in the control of circadian function but cannot infect visual circuits mediating visual perception or the reflex movement of the eyes. In this study, we used genetic complementation to test the hypothesis that gE and gI are required for entry into the specific retinal ganglion cells that project to visual centers. These data strongly suggest that gE and gI must function after the viruses enter primary neurons in the retina.

Animals↗
PubMed1993

Overall signal sequence hydrophobicity determines the in vivo translocation efficiency of a herpesvirus glycoprotein.

We have described three mutant strains of Pseudorabies virus that contain mutations in the signal sequence coding region of a nonessential envelope glycoprotein, gIII. The alterations disrupt, truncate, or eliminate the hydrophobic core domain of the signal sequence. Each mutant was assayed for its ability to promote the translocation of gIII across the endoplasmic reticulum membrane and the subsequent localization of the mature form of the glycoprotein to the infected cell surface or the virus envelope. Our results confirm and extend findings in other systems that the overall hydrophobicity of the signal sequence core region is a major determinant of translocation efficiency. We were unable to correlate simply the length of the core or the average hydrophobicity of core residues with export efficiency. Because our work involved the use of infectious virus mutants, we were able to identify a virus defect associated with a complete block in gIII export. This defect will facilitate a pseudo-reversion analysis of gIII signal sequence function.

Amino Acid Sequence↗
PubMed1993

Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function.

Animals↗
PubMed1993

Spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus.

We examined the responses of astrocytes, ramified microglia, and brain macrophages to CNS neuronal infection with virulent or attenuated strains of a swine alpha herpesvirus (pseudorabies virus, PRV). After PRV inoculation of the rat stomach or pancreas, the temporal course of viral replication and induced pathology of infected neurons were assessed in the dorsal motor nucleus of the vagus (DMV) and amygdala using an antiserum generated against PRV. Specific monoclonal antibodies against glial fibrillary acidic protein (GFAP), OX42, and ED1 and morphological criteria were used to classify non-neuronal cells. Both PRV strains infected DMV and motor neurons and then passed transneuronally to infect brainstem neurons that innervate the DMV. However, the onset of neuronal infection produced by the attenuated strain occurred approximately 20 hr later than infection with the virulent strain. Animals infected with the attenuated strain also survived longer, permitting transneuronal passage of virus into forebrain areas of the visceral neuraxis. Neuronal infection with both PRV strains produced consistent alterations in astrocytes, ramified microglia, and brain macrophages that correlated spatially and temporally with progressive stages of viral replication and neuronal pathology. Early stages of infection were characterized by increases in immunoreactivity for astrocytic GFAP and microglial OX42 that preceded overt signs of neuronal pathology. At later stages, GFAP immunoreactivity decreased dramatically in focal areas of neuronal infection while OX42 immunoreactivity continued to increase. Subsequently, ED1-immunoreactive brain macrophages infiltrated these infected areas. Double immunocytochemical labeling demonstrated that some astrocytes and brain macrophages were immunopositive for viral antigens but ramified microglia were not. The responses of glia and brain macrophages are consistent with a proposed role in restricting extracellular spread of virus by isolating or phagocytosing infected cells. These phenomena may contribute to the specific transneuronal transport exhibited by PRV.

Animals↗
PubMed1993

Pseudorabies virus infection of the rat central nervous system: ultrastructural characterization of viral replication, transport, and pathogenesis.

Pseudorabies virus (PRV) has been used extensively to map synaptic circuits in the CNS and PNS. A fundamental assumption of these studies is that the virus replicates within synaptically linked populations of neurons and does not spread through the extracellular space or by cell-to-cell fusion. In the present analysis we have used electron microscopy to characterize pathways of viral replication and egress that lead to transneuronal infection of neurons, and to document the non-neuronal response to neuronal infection. Three strains of PRV that differ in virulence were used to infect preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). The data demonstrate that viral replication and transneuronal passage occur in a stepwise fashion that utilizes existing cellular processes, and that the non-neuronal response to infection serves to restrict nonspecific spread of virus by isolating severely infected neurons. Specifically, capsids containing viral DNA replicate in the cell nucleus, traverse the endoplasmic reticulum to gain access to the cytoplasm, and acquire a bilaminar membrane envelope from the trans cisternae of the Golgi. The outer leaf of this envelope fuses with the neuron membrane to release virus adjacent to axon terminals that synapse upon the infected cell. A second fusion event involving the viral envelope and the afferent terminal releases the naked capsid into the bouton. Systematic analysis of serial sections demonstrated that release of virus from infected neurons occurs preferentially at sites of afferent contact. Nonspecific diffusion of virus from even the most severely infected cells is restricted by astrocytes and other non-neuronal elements that are mobilized to the site of viral infectivity. The ability of glia and macrophages to restrict spread of virus from necrotic neurons is the product of (1) temporal differences in the mobilization of these cells to the site of infection, (2) differential susceptibility of these cells to PRV infection, and (3) abortive viral replication in cells that are permissive for infection. The findings provide further insight into the intracellular routes of viral assembly and egress and support the contention that transneuronal spread of virus in the brain results from specific passage of virions through synaptically linked neurons rather than through cell fusion or release of virus into the extracellular space.

Animals↗
PubMed1992

Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system.

We previously demonstrated that intraocular injections of virulent and attenuated strains of pseudorabies virus (PRV) produce transneuronal infection of functionally distinct central visual circuits in the rat. The virulent Becker strain of PRV induces two temporally separated waves of infection that ultimately target all known retinorecipient neurons; the attenuated Bartha strain only infects a functionally distinct subset of these neurons. In this study, we demonstrate that deletion of a single viral gene encoding glycoprotein gI is sufficient to reproduce both the novel pattern of infectivity and the reduced neurovirulence of the Bartha strain of PRV. Glycoprotein gIII, a major viral membrane protein required for efficient adsorption of virus in cell culture, has no obvious role in determining the pattern of neuronal infectivity, but appears to function with gI to influence neurovirulence. These data suggest that neuroinvasiveness and virulence are the products of an interaction of viral envelope glycoproteins with as yet unidentified cellular receptors.

Animals↗
PubMed1992

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.

Animals↗
PubMed1991

Two alpha-herpesvirus strains are transported differentially in the rodent visual system.

Uptake and transneuronal passage of wild-type and attenuated strains of a swine alpha-herpesvirus (pseudorabies [PRV]) were examined in rat visual projections. Both strains of virus infected subpopulations of retinal ganglion cells and passed transneuronally to infect retino-recipient neurons in the forebrain. However, the location of infected forebrain neurons varied with the strain of virus. Intravitreal injection of wild-type virus produced two temporally separated waves of infection that eventually reached all known retino-recipient regions of the central neuraxis. By contrast, the attenuated strain of PRV selectively infected a functionally distinct subset of retinal ganglion cells with restricted central projections. The data indicate that projection-specific groups of ganglion cells are differentially susceptible to the two strains of virus and suggest that this sensitivity may be receptor mediated.

Animals↗
PubMed1991

Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export.

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.

Amino Acid Sequence↗
PubMed1991

Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress.

In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting double-membraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.

Animals↗
PubMed1991

Overexpression in bacterial and identification in infected cells of the pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5.

The ICP18.5 gene (UL28) of herpes simplex virus type 1 is a member of a well-conserved gene family among herpesviruses and is thought to play a role in localization of viral glycoproteins. We have cloned, sequenced, and expressed the entire pseudorabies virus (PRV) ICP18.5 open reading frame in Escherichia coli as a Cro-ICP18.5 fusion protein. Rabbit antiserum against Cro-ICP18.5 immunoprecipitated a 79-kDa protein from PRV-infected cells as well as a 79-kDa protein from in vitro translation of a T7 RNA polymerase transcript of the ICP18.5 gene. ICP18.5 could be detected in infected cells by 2 h postinfection. Analysis by indirect immunofluorescence demonstrated that ICP18.5 became associated with the nucleus. Subcellular fractionation confirmed that ICP18.5 synthesized during a pulse-chase experiment appeared in the nuclear fraction with time and was stable for at least 2.5 h after synthesis. Pulse-chase analysis revealed that ICP18.5 was synthesized as a monomer during a 2-min pulse labeling but formed faster sedimenting complexes which were sensitive to sodium dodecyl sulfate (SDS) treatment. The majority of ICP18.5 appeared in complexes with an antigenically unrelated 70-kDa protein. Immunoblot analysis of total infected-cell extracts using polyvalent anti-ICP18.5 serum demonstrated that a 74-kDa cellular protein in addition to the 79-kDa ICP18.5 was detected. This cellular protein was present at similar levels in uninfected cells and in PRV-infected cells at least 12 h into the infectious cycle.

Amino Acid Sequence↗
PubMed1991

The gIII glycoprotein of pseudorabies virus is involved in two distinct steps of virus attachment.

The entry of herpesviruses into cells involves two distinct stages: attachment or adsorption to the cell surface followed by internalization. The virus envelope glycoproteins have been implicated in both stages. Pseudorabies virus attaches to cells by an early interaction that involves the viral glycoprotein gIII and a cellular heparinlike substance. We examined the role of gIII in the attachment process by analysis of a set of viruses carrying defined gIII mutations. The initial attachment of gIII mutants with an internal deletion of 134 amino acids (PrV2) to MDBK cells was indistinguishable from that of wild-type virus. The adsorption of these mutants was, however, much more sensitive than that of wild-type virus to competing heparin. Furthermore, while attachment of wild-type virus to MDBK cells led to a rapid loss of sensitivity to heparin, this was not the case with PrV2, which could be displaced from the cell surface by heparin after it had attached to the cells. We conclude that glycoprotein gIII is involved in two distinct steps of virus attachment and that the second of these steps but not the first is defective in PrV2.

Adsorption↗
PubMed1990

The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus.

The pseudorabies virus gII gene shares significant homology with the gB gene of herpes simplex virus type 1. Unlike gB, however, gII is processed by specific protease cleavage events after the synthesis of its precursor. The processed forms are maintained in an oligomeric complex that includes disulfide linkages. In this report, we demonstrate the kinetics of modification, complex formation, and subsequent protease processing. In particular, we suggest that gII oligomer formation in the endoplasmic reticulum is an integral part of the export pathway and that protease cleavage occurs only after oligomers have formed. Furthermore, through the use of glycoprotein gene fusions between the gIII glycoprotein and the gII glycoprotein genes of pseudorabies virus, we have mapped a functional cleavage domain of gII to an 11-amino-acid segment.

Amino Acid Sequence↗