Biomedical subjects

L Haarr

Publications and source records attributed to L Haarr.

At least 19 recordsLinked to original sources

PubMed2005

Prevalence of, and risk factors for, HSV-2 antibodies in sexually transmitted disease patients, healthy pregnant females, blood donors and medical students in Tanzania and Norway.

The prevalence of specific HSV-2 antibodies was studied in Tanzanian and Norwegian sexually transmitted disease (STD) patients (1095) and non-STD patients (488). Correlates to demographic and behavioural factors were evaluated. Seropositivity was determined by the non-commercial peptide-55 enzyme-linked immunoassay. The prevalence of HSV-2 antibodies was 70% in Tanzanian and 17% in Norwegian STD patients, 35% in Tanzanian blood donors and pregnant women, and 4, 7 and 14% in Norwegian medical students, blood donors and pregnant women respectively. A higher HSV-2 prevalence was associated with female sex, increasing age, previous STDs, history of genital HSV infection, coitarchal age (age at first intercourse) <15 years and HIV seropositivity. Compared to previous data, the prevalence of HSV-2 antibodies in Tanzanian STD patients has increased remarkably. In Norwegian STD patients our results are consistent with, or lower than, the prevalence previously reported in Western Europe. Demographic rather than behavioural factors were associated with higher prevalence of HSV-2 antibodies in STD patients.

Adolescent↗

PubMed2001

Transcription from the gene encoding the herpesvirus entry receptor nectin-1 (HveC) in nervous tissue of adult mouse.

Both human and murine forms of nectin-1 (HveC, Prr1) can serve as entry receptors for several neurotropic herpesviruses, including herpes simplex viruses 1 and 2 (HSV-1, HSV-2), porcine pseudorabies virus (PRV), and bovine herpesvirus 1. HSV-1, HSV-2, and PRV can cause lethal neurological disease in mice whether inoculation is directly into the central nervous system or by peripheral routes. Expression of nectin-1 transcripts in cells of the adult mouse nervous system was assessed by in situ hybridization. Specific hybridization signals were detected in neurons in sensory, sympathetic, and parasympathetic ganglia of the peripheral nervous system. In addition, specific signals were observed in neurons of the ventral and dorsal horns of the spinal cord and of the brain stem, cerebellum, cerebral cortex, hippocampus, dentate gyrus, and olfactory bulb. These results show that the nectin-1 gene is widely transcribed in neurons in adult mouse. Nectin-1 is the only known receptor capable of mediating the entry of all three viruses, HSV-1, HSV-2, and PRV. Its pattern of expression in the nervous system suggests a key role in neurological disease caused by these viruses.

Animals↗

PubMed2000

A herpes simplex virus type 1 vector as marker for retrograde neuronal tracing: characterization of lacZ transcription and localization of labelled neuronal cells in sensory and autonomic ganglia after inoculation of the anterior segment of the eye.

Herpes simplex virus type 1 (HSV-1) is a human, neurotropic pathogen which also can infect experimental animals. Much interest has been focused on genetic modification of HSV-1 so that it can be used as a vector for gene delivery and for tracing neuronal connections. For expression of a foreign gene inserted into the HSV-1 genome, both the site of insertion and the promoter activity are important. We have used a previously described HSV-1 vector, KOS/58, to demonstrate that the beta-galactosidase gene inserted together with a neurofilament L promoter into the coding region of the glycoprotein C (gC) gene is under the control of the foreign promoter rather than under that of the gC gene. This was performed by isolation of RNA from infected, neuron-like PC12 cells and Northern blotting using probes from various regions of the modified part of the genome. The vector was then inoculated in the cornea, subconjunctivally, or into the anterior chamber of the mouse eye. Whole mounts of the trigeminal, superior cervical and pterygopalatine ganglions were stained for beta-galactosidase. The localization of labelled neurons was consistent with retrograde axonal transport as the principal way of neuronal infection indicating that KOS/58 could be used as a retrograde tracer. The position of the labelled cells suggests a somatotopic organization of the mouse trigeminal and superior cervical ganglion similar to that of rats and rabbits.

Animals↗

PubMed1999

Infection of rat brain cell aggregates with neurovirulent and nonneurovirulent strains of herpes simplex virus type 1.

Rat brain cell aggregates represent a three-dimensional tissue culture system of brain tissue in the form of small, multicellular spheroids. In the present work, we have infected these "minibrains" with neurovirulent, nonneurovirulent, and nonreplicating strains of HSV-1. The neurovirulent strains 17(+) and KOS(M) spread rapidly through the aggregates, while the nonreplicating ICP4 deletion mutant KD6 infected cells only at the periphery of the aggregates. Spread and replication of the nonneurovirulent strains RE6 and tk-7, and to some extent also of R13/1, were restricted. The interaction between different strains of HSV-1 and the rat brain cell aggregates is thus comparable to that seen in the brain, suggesting that the aggregates represent a useful tool for studying HSV-1 infection of brain tissue in vitro.

Animals↗

PubMed1998

Identification of the UL4 protein of herpes simplex virus type 1.

Herpes simplex virus type 1 gene UL4 is predicted to encode a 199-amino-acid protein with a molecular mass of 21 5 kDa. We report here identification of this protein and its localization in the nuclei of infected cells. Antisera raised against oligopeptides corresponding to the C terminus of the predicted UL4 protein were used for identification of a 25 kDa protein as the product of the UL4 gene. This protein was not detected in cells infected with a UL4 defective mutant virus, but was synthesized by coupled in vitro transcription-translation of the UL4 gene. Synthesis of the 25 kDa protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis, indicating that the UL4 gene is expressed with gamma kinetics. Subcellular fractionation showed the protein to be localized in the nucleus. It was not detected in virions or light particles.

Amino Acid Sequence↗

PubMed1998

The UL4 gene of herpes simplex virus type 1 is dispensable for latency, reactivation and pathogenesis in mice.

The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.

Amino Acid Sequence↗

PubMed1998

Prevalence of HSV-2 antibodies among STD clinic patients in Tanzania.

Patients attending a referral sexually transmissible diseases clinic at Muhimbili Medical Centre in Dar-es-Salaam during the period 1989 to 1993 were examined for herpes simplex virus type 2 (HSV-2) antibodies. An ELISA technique, using glycoprotein G of HSV-2 as antigen, was used to test 294 patients' sera. Of these, 126 sera were HSV-2 positive, while 168 were negative, yielding an overall HSV-2 prevalence of 42.9%. Sixty-three per cent of the women and 35.5% of the men were HSV-2 positive. Seropositivity rose from 8.7% in the youngest men to 61.5% in the oldest male age group, while even the youngest women aged 20 or less had an HSV-2 prevalence of 55.6%. There was a significant positive association between HIV and HSV-2 seropositivity (P=0.0006), most pronounced among the youngest women. There was no over-representation of HSV-2 positivity among patients with genital ulcer disease, indicating that other causes of this disease could be more common than HSV-2 in Tanzania.

Adult↗

PubMed1997

Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion.

Purification of herpes simplex virus type 1 (HSV-1) is often performed by centrifugation in gradients of various materials. A major problem with such procedures is a gradual decrease in the infectivity of the virus, probably due to the influence of the gradient material. In the present work we have compared Nycodenz gradients with Ficoll gradients for HSV-1 purification. Both gradient materials have low osmolarities. The purity of the preparations recovered from the two gradients was similar, as measured by electron microscopy and two-dimensional gel electrophoresis, and the yield of infectious virus was approximately the same. Szilágyi & Cunningham (22) reported previously that Ficoll gradients separate one band of complete virions and another band containing light particles devoid of nucleocapsids. In Nycodenz gradients we observed the same bands, and an additional third band which contained aggregates and disrupted particles. In this respect, the separation in Nycodenz is slightly superior to that in Ficoll. The purified HSV-1 virions were subjected to analytical differential centrifugation and an S-value of 670S was calculated for the virus.

Animals↗

PubMed1997

Analysis of the 2-kilobase latency-associated transcript expressed in PC12 cells productively infected with herpes simplex virus type 1: evidence for a stable, nonlinear structure.

The major latency-associated transcript (LAT) expressed in PC12 cells productively infected with herpes simplex virus type 1 is a 2-kb, nonpolyadenylated RNA molecule that accumulates in the nuclei of infected cells. In actinomycin D-treated cells, the 2-kb LAT gene transcript has a half-life considerably greater than 12 h. After polyacrylamide gel electrophoresis, two species of the transcript were observed, a major species that was retarded in the gel and a minor species that migrated as a 1.96-kb RNA molecule. RNase H digestion after hybridization of the RNA with an oligonucleotide complementary to positions -80 to -101 relative to the 3' end of the 2-kb LAT gene transcript changed the mobility of the retarded species into that of the rapidly migrating species. Our data indicate that the 2-kb LAT gene transcript expressed in productively infected PC12 cells is present in a stable, nonlinear form.

Animals↗

PubMed1996

Two-dimensional gel analysis of [35S]methionine labelled and phosphorylated proteins present in virions and light particles of herpes simplex virus type 1, and detection of potentially new structural proteins.

Cells infected with herpes simplex virus (HSV) synthesize both infectious viruses and non-infectious light particles (L-particles). The latter contain the envelope and tegument components of the virions, but lack virus capsid and DNA. Electrophoresis in SDS-polyacrylamide gels (SDS-PAGE) has been used extensively for analysis of structural proteins in virions and L-particles. Two-dimensional (2-D) gel electrophoresis, however has a markedly higher resolution, and in the present work we have used this technique to study both [35S]methionine labelled and phosphorylated structural proteins in virions and L-particles. Proteins were assigned to the tegument or the envelope by the analysis of L-particles. Localization of structural proteins was also determined by stepwise solubilization in the presence of the neutral detergent NP-40 and NaCl, and by isolation of capsids from nuclei of infected cells. Different steps in posttranslational modification can be detected by 2-D gel electrophoresis such that a single polypeptide may appear as several spots. This was most clearly observed for some of the HSV-encoded glycoproteins which were shown to exist in multiple forms in the virion. Some polypeptides apparently not identified previously were either capsid associated, or localized in the tegument or envelope. The degrees of phosphorylation in L-particles and virions are almost identical for some proteins, but markedly different for others. Thus, glycoprotein E of HSV-1 is for the first time shown to be phosphorylated, and most heavily so in virions. The IE VMW)110 protein represents a group of proteins which are more phosphorylated in L-particles than in virions. Attempts are made to correlate the proteins detected by 2-D analysis with those previously separated by SDS-PAGE.

Animals↗

PubMed1996

Human interferon reduces surface expression but not total production of herpes simplex virus type 1 glycoproteins gC and gE in heterologous hamster cells.

The effect of interferon (IFN) on herpes simplex virus type 1 (HSV-1)-induced glycoproteins gC and gE was investigated in a heterologous IFN/cell model. In this model, the effect on surface expression of the glycoproteins could be studied separately from the effect on virus multiplication. Pretreatment of baby hamster kidney cells (BHK) with heterologous human leukocyte IFN suppressed surface expression of HSV-1-encoded gC and gE but had no influence on total production of the glycoproteins. This was in contrast to the effect on human embryonic fibroblast cells (HE) (homologous IFN and cells), where surface expression as well as total production of glycoproteins were reduced. The surface expression was demonstrated by antibody-sensitized monodisperse polystyrene beads, and immunoblotting and two-dimensional electrophoretic analysis of radioisotope-labeled proteins were used to study the total production.

Animals↗

PubMed1995

Labeling of surface proteins of herpes simplex virus type 1 using a modified biotin-streptavidin system.

Methods of labeling surface proteins on herpes simplex virus (HSV) which have minimal effect on the biological activity of the virus are useful for the study of both the localization and function(s) of surface proteins. The present work describes a procedure using a water-soluble biotin compound, sulfo-NHS-biotin, which is unable to penetrate biological membranes and reacts with primary amines in proteins. Labeled proteins were detected by binding of [125I]streptavidin. Specific reaction with surface proteins was shown in Western blots using antibodies against selected proteins in the envelope or in the tegument. Proteins susceptible to iodination were also biotinylated, but the efficiency of labeling varied from one protein to another. As a result of freezing and thawing of the virus, as well as the manipulations involved in Ficoll gradient purification, internal proteins were labeled. The infectivity of the virus was reduced by approximately 40% after biotinylation. Labeled viruses were visualized by fluorescein isothiocyanate-conjugated streptavidin, and seen as distinct spots on the surface of the cells.

Animals↗

PubMed1994

The herpes simplex virus type 1 particle: structure and molecular functions. Review article.

This review is a summary of our present knowledge with respect to the structure of the virion of herpes simplex virus type 1. The virion consists of a capsid into which the DNA is packaged, a tegument and an external envelope. The protein compositions of the structures outside the genome are described as well as the functions of individual proteins. Seven capsid proteins are identified, and two of them are mainly present in precursors of mature DNA-containing capsids. The protein components of the 150 hexamers and 12 pentamers in the icosahedral capsid are known. These capsomers all have a central channel and are connected by Y-shaped triplexes. In contrast to the capsid, the tegument has a less defined structure in which 11 proteins have been identified so far. Most of them are phosphorylated. Eleven virus-encoded glycoproteins are present in the envelope, and there may be a few more membrane proteins not yet identified. Functions of these glycoproteins include attachment to and penetration of the cellular membrane. The structural proteins, their functions, coding genes and localizations are listed in table form.

Capsid↗

PubMed1993

[Human herpesvirus 6].

Human herpesvirus-6 (HHV-6) is the most recently discovered herpesvirus. It shares several characteristic properties with the other herpesviruses; the genome is large, consisting of approximately 170,000 base pairs and potentially containing more than 70 genes. The virus also probably forms latent infections in the host. It is widely distributed in the population, and seropositivity reaches 60-90% by the age of two. HHV-6 is associated with the childhood disease exanthema subitum, and probably also causes heterophile antibody-negative mononucleosis. Our present knowledge about the virus is reviewed.

Herpesviridae Infections↗

PubMed1993

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption.

Animals↗

PubMed1990

Identification and characterization of the virion protein products of herpes simplex virus type 1 gene UL47.

We have identified two encoded proteins with an antiserum raised against a synthetic oligopeptide corresponding to amino acids 671 to 684 of the predicted protein product of gene UL47 of herpes simplex virus type 1 (HSV-1). They have apparent Mrs of 82,000 and 81,000 and are both major virion components located in the tegument. The 82/81K proteins were first detected in infected cells in minor amounts 6 h after infection at 37 degrees C but were later (from 10 h until 24 h after infection) present in large amounts. UL47 regulation was investigated using phosphonoacetic acid (PAA), an inhibitor of DNA synthesis: the amounts of the 82/81K protein synthesized were compared with those of 65KDBP, an early gene product, and 21K/22K, a true late gene product. The data showed that UL47 is regulated as a true late gene.

Amino Acid Sequence↗

PubMed1990

Localization on the herpes simplex virus type 1 genome of a region encoding proteins involved in adsorption to the cellular receptor.

We have previously shown that aminoglycosides such as neomycin and the polyamino acids polylysine and polyarginine selectively inhibit the binding of herpes simplex virus type 1 (HSV-1) to the cellular receptor, whereas HSV-2 infection is unaffected. In the present study we took advantage of this difference between HSV-1 and HSV-2 by using HSV(-1)-HSV(-2) intertypic recombinants to locate a region on the HSV-1 genome encoding proteins affecting the binding of the virion to the cellular receptor. The results were consistent with those obtained by marker rescue experiments. The identified region, which mapped between coordinates 0.580 and 0.687, contains two partial and eight complete genes, including the glycoprotein C (gC) gene and two others with potential transmembrane sequences. Various gC monoclonal antibody-resistant mutants of HSV-1 as well as a mutant completely lacking gC were found to be fully sensitive to neomycin, suggesting that gC is not the site of drug sensitivity and is not essential for adsorption of virus to the cellular receptor. However, the rate of adsorption was reduced in the absence of gC, indicating a facilitating function of the glycoprotein. The universal nature of this HSV-1 receptor binding was revealed by the similarity in drug sensitivity of infectivity in four different cell lines from various tissues and species.

Adsorption↗

PubMed1989

Herpes simplex virus-1-specific proteins are involved in alteration of polyphosphoinositide metabolism in baby-hamster kidney cells.

Herpes simplex virus type 1 (HSV-1) induces altered phosphoinositide metabolism in baby hamster kidney (BHK) cells, measured as incorporation of [3H]inositol or [32P]Pi [Langeland, Haarr & Holmsen (1986) Biochem. J. 237, 707-712]. We now report that this response in the inositol phospholipids is dependent on virus-specific proteins synthesized in the beta (early) stage of virus protein synthesis. This was demonstrated both by resistance to the inhibitory effect of cycloheximide after this stage of infection, and by the use of temperature-sensitive (ts) mutants of HSV-1; ts mutants in which protein synthesis was blocked so that only the alpha proteins were expressed showed a PIP2/PIP (phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 4-monophosphate) ratio similar to uninfected cells, while ts mutants which were defective in protein synthesis at a late beta stage or later showed increased PIP2/PIP ratios similar to cells infected by wild type HSV-1.

Animals↗