Expression of entry receptor nectin-1 of herpes simplex virus 1 and/or herpes simplex virus 2 in normal and neoplastic human nervous system tissues

Herpes simplex virus 1 and/or Herpes simplex virus 2 (HSV) are important pathogens of human nervous system (NS) and genetically modified HSV strains have been proposed as vectors for gene therapy targeting the brain and brain tumors. Nectin-1 is an immunoglobulin-like adhesion molecule that participates in the formation of synapses and serves as an entry receptor for HSV. The expression pattern of nectin-1 in normal human NS and brain tumors is not well understood. To better understand the nectin-1 expression in normal and neoplastic human NS, immunohistochemistry was used to detect the nectin-1 expression in sections of normal human brain, spinal cord and trigeminal and dorsal root ganglia (n=10) and in sections of primary NS neoplasms (n=22). In normal human NS, nectin-1 was detected in the soma and processes of central and peripheral neurons, in ependymal cells, choroid plexus epithelial cells, vascular endothelial cells and meningothelial cells. Oligodendrocytes, astrocytes, vascular smooth muscle cells, and Schwann cells showed variable immunoreactivity. Among tumors, schwannoma, fibrous meningioma, and medulloblastoma were nectin-1 negative. Oligodendroglioma, ependymoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme and meningothelial meningioma showed weak focal nectin-1-positivity. Ganglion cells of ganglioglioma were strongly positive. These studies provide novel information about the expression of nectin-1 in normal and neoplastic NS, and thus may lead to a better understanding of cell targeting by HSV during HSV-induced neurological disease and during a HSV-based gene therapy.     

Latent herpes simplex virus type 1 in normal and Alzheimer's disease brains.

A viral aetiology has long been suspected for Alzheimer's disease (AD) but until now, techniques have not been sufficiently sensitive to provide clear evidence for or against the presence of any viral genome in AD brain. We have used the very highly sensitive method of polymerase chain reaction to look for herpes simplex virus type 1 (HSV1) DNA, specifically the viral thymidine kinase (TK) gene, in autopsy brain specimens. DNA-samples from HSV-infected and uninfected Vero cells have been examined concurrently to provide standard "HSV-positive" and "HSV-negative" samples, the latter guarding also against false positives caused by cross-contamination. To preclude false negatives, we have checked the presence of the human gene, hypoxanthine phosphoribosyl transferase. In all specimens from 8 AD patients and 6 normal individuals (temporal, frontal and hippocampal), we have found viral TK sequences. In contrast, in preliminary studies on lymphocytes from normals and AD patients, we did not find TK sequences. It is postulated that factors such as number or expression of viral genes and host susceptibility might be related to incidence of AD.     

The requirements for herpes simplex virus type 1 cell-cell spread via nectin-1 parallel those for virus entry

Herpes simplex virus type 1 (HSV-1) spreads from an infected cell to an uninfected cell by virus entry, virus-induced cell fusion, and cell-cell spread. The three forms of virus spread require the viral proteins gB, gD, and gH-gL, as well as a cellular gD receptor. The mutual requirement for the fusion glycoproteins and gD receptor suggests that virus entry, cell fusion, and cell-cell spread occur by a similar mechanism. The goals of this study were to examine the role of the nectin-1alpha transmembrane domain and cytoplasmic tail in cell-cell spread and to obtain a better understanding of the receptor-dependent events occurring at the plasma membrane during cell-cell spread. We determined that an intact nectin-1alpha V-like domain was required for cell-cell spread, while a membrane-spanning domain and cytoplasmic tail were not. Chimeric forms of nectin-1 that were non-functional for virus entry did not mediate cell-cell spread regardless of whether they could mediate cell fusion. Also, cell-cell spread of syncytial isolates was dependent upon nectin-1alpha expression and occurred through a nectin-1-dependent mechanism. Taken together, our results indicate that nectin-1-dependent events occurring at the plasma membrane during cell-cell spread were equivalent to those for virus entry.     

In situ detection of DNA-binding proteins in herpes simplex virus type 1-infected cells

An in situ assay for detecting DNA-binding proteins in herpes simplex virus type 1 (HSV-1)-infected cells is described. Seventeen HSV-induced DNA-binding species were visible with nicked, double-stranded DNA as a substrate, while fourteen virus-induced DNA-binding fractions were present in gels containing nuclease-treated, single-stranded DNA. The effects of HSV on cellular DNA-binding protein expression could also be seen. The resolution of DNA-binding fractions was dependent upon the type of DNA substrate utilized, high salt extraction of DNA-binding components and their physical separation from infected cell DNAs, dialysis of the high salt and the length of DNase treatment of gels following electrophoresis.     

Polypeptide synthesized in herpes simplex virus type 2-infected HEp-2 cells.

The proteins induced by herpes simplex virus type 2 in infected HEp-2 cells were studied by high-resolution polyacrylamide slab-gel electrophoresis. A total of 51 infected cell-specific polypeptides (ICSP) were detected, accounting for three-fourths of the coding capacity of the virus. The induced polypeptides were allocated to three groups based on their time of synthesis in the virus growth cycle. Cycloheximide treatment of infected cells during the early stage of virus growth was found to cause irreversible inhibition of protein synthesis. Herpes simplex virus type 2 polypeptides which underwent considerable posttranslational modification were detected.     

High seroprevalence of herpes simplex virus type 2 infection in French human immunodeficiency virus type 1-infected outpatients

Using commercially available herpes simplex virus (HSV) type-specific serological diagnostic tests, HSV type 2 (HSV-2) antibody prevalence was assessed in two parallel prospective studies including 534 human immunodeficiency virus type 1 (HIV-1)-infected outpatients living in two areas of northern France. In the first cohort of 434 subjects, 223 (51%) individuals demonstrated a positive HSV-2 serological status while 66 (66%) of 100 subjects in the second cohort were seropositive for HSV-2 (51 versus 66%; P = 0.08). Among the 223 HSV-2-seropositive subjects identified in the first study cohort, only 22 (10%) had suffered from recurrent anogenital lesions during the past 12 months while 154 (69%) had no clinical history of herpesvirus infection. Our findings demonstrate high proportions of subclinical and undiagnosed HSV-2 infection in HIV-1-infected individuals and suggest that HSV type-specific serological testing in the French HIV-1-infected subpopulation could be an efficient strategy to diagnose clinically asymptomatic HSV-2 infections.     

Glycoprotein D homologs in herpes simplex virus type 1, pseudorabies virus, and bovine herpes virus type 1 bind directly to human HveC(nectin-1) with different affinities

Distinct subsets of human receptors for alphaherpesviruses mediate the entry of herpes simplex virus (HSV), pseudorabies virus (PrV), or bovine herpes virus type 1 (BHV-1) into cells. Glycoprotein D (gD) is essential for receptor-mediated entry of all three viruses into cells. However, the gD homologs of these viruses share only 22-33% amino acid identity. Several entry receptors for HSV have been identified. Two of these, HveA (HVEM) and HveC (nectin-1), mediate entry of most HSV-1 and HSV-2 strains and are bound directly by HSV gD. A third receptor, HveB (nectin-2), mediates entry of HSV-2 and only a limited number of HSV-1 strains. HveB and HveC can also serve as entry receptors for PrV, whereas only HveC can serve this function for BHV-1. We show here that gD from PrV and BHV-1 binds directly to the human receptors that mediate PrV and BHV-1 entry. We expressed soluble forms of PrV gD and BHV-1 gD using recombinant baculoviruses and purified each protein. Using ELISA, we detected direct binding of PrV gD to HveB and HveC and direct binding of BHV-1 gD to HveC. Biosensor analysis revealed that PrV gD had a 10-fold higher affinity than HSV-1 gD for human HveC. In contrast, the binding of BHV-1 gD to HveC was weak. PrV gD and HSV-1 gD competed for binding to the V domain of HveC and both inhibited entry of the homologous and heterologous viruses. These data suggest that the two forms of gD bind to a common region on human HveC despite their low amino acid similarity. Based on affinities for human HveC, we predict a porcine HveC homolog may be important for PrV infection in its natural host, whereas a BHV-1 infection in its natural host may be mediated by a receptor other than a bovine HveC homolog.     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

Hemagglutination by herpes simplex virus type 1

Summary The McIntyre and HSZP strains as well as clinical isolate of herpes simplex virus type 1 were found to agglutinate C57B1/10su and CBA mouse red blood cells. The hemagglutinating activity was inhibited by antisera that neutralized the infectivity of the virus.     

Expression and characterization of baculovirus expressed herpes simplex virus type 1 glycoprotein L

Summary We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26–27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer <1: 10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.     

Some structural antigens of herpes simplex virus type 1.

Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.     

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.     

Interference between strains of type 1 and type 2 herpes simplex virus.

Herpes simplex virus type 2 (HSV-2) markedly interferes with the replication of herpes simplex virus type 1 (HSV-1) upon simultaneous infection of HEp-2 cells, even under conditions where HSV-1 infection precedes that of HSV-2 by 3 hr. Interference required infective HSV-2 virus. The HSV-1 progeny of mixed infections were found to be phenotypically mixed. In most cases, prior infection with homologous strains slightly inhibits the replication of a superinfecting HSV-1 strain. An exception to this finding is HSV-1 strain MP, whose replication is accelerated by prior infection with nondefective HSV-1 strains. The implications of these findings as related to studies of defective HSV, to studies of the genetic interactions between HSV-1 and HSV-2, and to clinical infections with HSV are discussed.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Inhibitory effect of herpes simplex virus type 1 on type 2 virus replication.

Simultaneous infection with herpes simplex type I and type 2 viruses of chick embryo fibroblasts (CEF), which are only permissive for type 2 virus, or rabbit embryo fibroblasts (REF), which are permissive for both virus types, resulted in a marked reduction of type 2 virus production. This effect was dependent on the m.o.i. of type I, being expressed at a high rather than a low m.o.i. The rate of interference decreased with the prolongation of the interval between infection with type 2 and type I viruses. No evidence suggestive of interferon involvement was obtained. Partial inactivation of type 2 virus by ultraviolet irradiation enhanced the inhibitory effect of type I virus. On the other hand, u.v. irradiation of type I virus resulted in a progressive loss of inhibitory activity. The results of the present experiments suggest that a type I genome function is responsible for the interfering effect, and that an early step in the growth of type 2 virus is sensitive to the particular type I virus product involved.     

Complement-fixing antibody to the AG-4 antigen in herpes simplex virus type 2-infected patients.

Sera collected from confirmed herpes simplex virus type 2 (HSV-2) patients were found to be devoid of complement-fixing antibody to the AG-4 antigen at the time of the herpes lesion outbreak in 10 out of 13 cases. However, 1 to 4 weeks after HSV-2 lesion appearance, 28 out of 30 patients acquired complement-fixing antibody to the AG-4 antigen. The sera of these patients contained immunoglobulin M antibody activity and the ability to immunoprecipitate a 160,000-molecular weight early HSV-2 antigen (the AG-4 antigen). Also, these sera were used to show that a variety of anti-herpes virus compounds had a negligible effect on AG-4 production in HSV-2-infected HEp-2 cells. The majority of the compounds tested (including acycloguanosine and phosphonoformate) are known to inhibit late antigen production, suggesting that the AG-4 antigen is an early antigen. It is probably an immediate early antigen (alpha) as it is formed in the presence of cycloheximide and actinomycin D, a treatment which is used to accumulate alpha proteins.     

Efficacy of herpes simplex virus type 1 immunization in protecting against acute and latent infection by herpes simplex virus type 2 in mice.

ICR mice were immunized with herpes simplex virus type 1 (HSV-1) and later challenged with HSV-2 by footpad inoculation. Both immunized animals and age-matched, nonimmunized controls were observed for ascending neurological disease and latent infection of spinal ganglia resulting from the HSV-2 challenge. Control animals had a 78% incidence of acute and latent infection compared with a 1.7% incidence in immunized mice. The data show immunity to HSV-1 is protective against both acute and latent infection by HSV-2.     

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1.

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2 infected LL-1 cells demonstrated a block in HSV-2-specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSV-specific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.