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B Wigdahl

Publications and source records attributed to B Wigdahl.

At least 37 records · Page 2Linked to original sources
PubMed1994

Neuroglial-specific factors and the regulation of retrovirus transcription.

Retroviruses have been implicated as causative agents of a variety of human diseases including malignancy, immune system dysfunction, and neurologic disorders. Despite the isolation of various retroviral agents from patients suffering from malignant neoplasias and neurologic disorders, only the human T-cell lymphotropic virus type I (HTLV-I) and the human immunodeficiency virus (HIV) have been definitively accepted as etiologic agents of human disease (Hjelle, 1991; Gessain and Gout, 1992; Rosenblatt, 1993). Because of their increasingly defined roles in disease progression, the replication of HTLV-I and HIV is an important focus for understanding the pathogenic processes resulting from viral infection. Of particular interest are the molecular mechanisms by which expression of retroviral genomes is regulated by their regulatory units, the long terminal repeats (LTR), in a manner specific to the cellular targets which they infect.

AIDS Dementia Complex↗
PubMed1994

Identification of human T-cell lymphotropic virus type I 21-base-pair repeat-specific and glial cell-specific DNA-protein complexes.

The human T-cell lymphotropic virus type I (HTLV-I)-encoded protein, Tax, is capable of trans-activating HTLV-I transcription by interacting with specific sequences in the HTLV-I long terminal repeat (LTR) which comprise an inducible enhancer containing three imperfect tandem repeats of a 21-bp sequence. There is no evidence that purified Tax can bind to DNA in the absence of cellular factors, suggesting that Tax most likely regulates transcription via interaction with cellular factors. Since HTLV-I is a documented agent of adult T-cell leukemia and tropical spastic paraparesis, disorders of the immune and nervous systems, respectively, characterization of cellular factors of lymphoid and neuroglial origin which interact with the 21-bp repeat elements is essential to understanding of the mechanisms involved in basal and Tax-mediated transcription in cells of immune and nervous system origin. Utilizing electrophoretic mobility shift (EMS) analyses, we have detected both 21-bp repeat-specific and glial cell-specific DNA-protein complexes. Several 21-bp repeat-specific DNA-protein complexes were detected when nuclear extracts derived from cells of lymphoid (Jurkat, SupT1, and H9), neuronal (IMR-32 and SK-N-MC), and glial (U-373 MG, Hs683, and U-118) origin were used in reactions with each of the three 21-bp repeat elements. In addition, a glial cell-specific DNA-protein complex was detected when nuclear extracts derived from U-373 MG, Hs683, and U-118 glial cell lines reacted with the promoter-distal and central 21-bp repeat elements. Furthermore, EMS analyses performed with nuclear extracts derived from lymphocytic and glial cell origin and a 223-bp fragment of the HTLV-I long terminal repeat encompassing the three 21-bp repeat elements (designated Tax-responsive elements 1 and 2, TRE-1/-2) have also resulted in the detection of glial cell type-specific DNA-protein complexes. Competition EMS analyses with oligonucleotides containing transcription factor binding site sequences indicate the involvement of a cyclic AMP response element binding protein in the formation of DNA-protein complexes which form with all three 21-bp repeat elements and the glial cell-specific DNA-protein complex as well as the involvement of Sp1 or an Sp1-related factor in the formation of the 21-bp repeat III-specific DNA-protein complexes.

Base Sequence↗
PubMed1994

Identification of HTLV-I 21 bp repeat-specific DNA-protein complexes.

The human T cell lymphotropic virus type 1 (HTLV-I)-encoded protein, Tax, is capable of transcriptionally trans-activating HTLV-I by interacting with specific sequences in the HTLV-I long terminal repeat (LTR) which comprise an inducible enhancer containing three imperfect tandem repeats of a 21 bp sequence. Evidence suggests that Tax is incapable of directly interacting with DNA; therefore, Tax most likely regulates transcription via interaction with cellular factors. In addition to a role in Tax-mediated trans-activation, cellular factors are also critical elements in basal HTLV-I LTR-directed transcription. Therefore, characterization of cellular factors which interact with the 21 bp repeat elements is essential to understanding the molecular mechanisms involved in both basal and Tax-mediated transcription from the HTLV-I LTR. Utilizing electrophoretic mobility shift (EMS) analyses, we have detected 21 bp repeat-specific DNA-protein complexes when nuclear extracts derived from cells of lymphoid (Jurkat, SupT1, and H9) and monocytoid (U937) origin were reacted with each of the three 21 bp repeat elements. Furthermore, results from EMS competition analyses utilizing unlabeled 21 bp repeats as competitor DNAs have indicated a difference in the ability of each unlabeled 21 bp repeat to compete for the specific DNA-protein complexes formed between the nuclear extracts and radiolabeled 21 bp repeats. In each case, the most effective competitor was the homologous, unlabeled 21 bp repeat element. These results demonstrate that there are 21 bp repeat-specific DNA-protein complexes and suggest functional differences among the three 21 bp repeat elements.

Base Sequence↗
PubMed1992

Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0.

Dominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.

Antiviral Agents↗
PubMed1992

Identification of dominant-negative mutants of the herpes simplex virus type 1 immediate-early protein ICP0.

ICP0 is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICP0 mediates transactivation.

Animals↗
PubMed1992

Effect of gamma-interferon on the expression of major histocompatibility complex class I and II gene products in neural cells isolated from the developing human fetal peripheral nervous system.

The effect of gamma-interferon (gamma-IFN) on the expression of major histocompatibility complex (MHC) gene products was examined in the developing human fetal peripheral nervous system. RNA blot hybridization analysis of total RNA isolated from human fetal dorsal root ganglia (DRG) neural cell populations cultured in vitro for 5 days resulted in the detection of both MHC class I- and class II-specific RNAs. As determined by protein immunoblotting and fluorescence-activated flow cytometry, MHC class I and II proteins were also readily detectable in cultured human fetal DRG neural cell populations 5 days after isolation. In addition, treatment of 3-day human fetal DRG neural cells with gamma-IFN (100 U/ml; 48 h) resulted in a marked increase in the level of MHC class I- and class II-specific RNA and protein without inhibiting the proliferation of the neural cell population. These results suggest that changes in the levels of selected cytokines such as gamma-IFN may alter the ability of specific neural cell populations present in the developing human nervous system to participate in immune reactions by alteration of MHC class I and II antigen expression which may lead to perturbation in glial cell function and ultimately to nervous system dysfunction in general.

Cell Division↗
PubMed1991

Maintenance of human immunodeficiency virus type-1 proviral DNA in human fetal dorsal root ganglia neural cells following a nonproductive infection.

Infection of the nervous system by human immunodeficiency virus type-1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome (AIDS)-associated neurologic dysfunction and direct infection of glia has been suggested as one of the potential mechanisms leading to deterioration of nervous system function. We have been examining the interaction of HIV-1 with the developing peripheral nervous system in vitro, and have previously shown that HIV-1 infection of primary human fetal dorsal root ganglia (DRG) neural cells resulted in HIV-1 gag antigen expression in approximately 70% of the glial cell subpopulation with little, if any, cytopathic damage to the infected cells. Accumulation of HIV-1 gag antigens and viral mRNA reached a maximum by 2-3 days postinfection and declined thereafter to minimally detectable levels in the surviving neural cell population. In addition, infection of the fetal DRG neural cells appeared to be abortive or nonproductive, with little if any, infectious progeny virus production. However, we have been able to detect HIV-1-specific proviral DNA as late as 24 days postinfection by polymerase chain reaction amplification and subsequent DNA blot hybridization. These results suggest that accumulation of HIV-1 structural proteins without the assembly and release of mature virus in HIV-1-infected human fetal DRG neural cells results in a nonproductive infection and maintenance of HIV-1 proviral DNA in the infected cell population.

Cell Transformation, Viral↗
PubMed1990

Analysis of nonproductive human immunodeficiency virus type 1 infection of human fetal dorsal root ganglia glial cells.

Direct infection of glia by human immunodeficiency virus type 1 (HIV-1) has been suggested as one of several mechanisms responsible for the severe neurologic complications observed in both neonates and adults with the acquired immunodeficiency syndrome. We have demonstrated by protein immunoblotting analysis that HIV-1 infection of human fetal glial cells isolated from the dorsal root ganglia (DRG) of the developing human peripheral nervous system results in viral gag antigen expression with little, if any, detectable env gene products. No cytopathogenicity was evident in the infected cell population. Blot hybridization analyses indicate transient expression of the HIV-1 genome with maximum levels of virus-specific RNA being observed between 2 and 3 days postinfection and decreasing below the limits of detection by 16 days postinfection. To determine whether infection of the human fetal DRG glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase assays were performed. Direct assay of HIV-1-infected neural cell supernatants as well as exposure of permissive SupT1 cells to these HIV-1-infected neural cell supernatants resulted in no demonstrable reverse transcriptase activity in either the HIV-1-infected DRG glial cell supernatants or the SupT1 cell supernatants. Although transmission electron microscopy analyses have suggested the absence of intracellular viral particles, highly electron-dense inclusions in the cytoplasm of HIV-1-infected DRG glial cells were observed. The nature of the intracellular cytoplasmic inclusions is under current investigation. Cumulatively, these data suggest that the interaction of HIV-1 with human fetal DRG neural cells results in transient expression of the HIV genome culminating in a nonproductive infection.

Cells, Cultured↗
PubMed1990

Analysis of major histocompatibility complex gene products in tissues isolated from the developing human nervous system.

We have examined the expression of the major histocompatibility complex (MHC) class I and II gene products in the developing human fetal peripheral nervous system. As determined by RNA blot hybridization analysis, MHC class I RNA was readily detectable in extracts prepared from dorsal root ganglia (DRG) obtained from aborted human fetal material. However, utilizing similar methodology, it was not possible to detect MHC class II RNA. In conjunction with these studies, expression of MHC class I and II proteins in primary human fetal DRG tissue was examined by fluorescence-activated flow cytometry and protein immunoblotting. Consistent with the detection of MHC-specific RNA, the accumulation of MHC class I-specific protein was readily detectable in human fetal DRG neural cell populations with little, if any, accumulation of MHC class II-specific protein evident. These studies suggest that MHC gene products may be expressed early in the development of the human nervous system resulting in the generation of specific immunocompetent neural cell populations.

Flow Cytometry↗
PubMed1989

Transient expression of human immunodeficiency virus type 1 genome results in a nonproductive infection in human fetal dorsal root ganglia glial cells.

Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), has been implicated in the generation of AIDS-associated neurologic dysfunction. We are currently examining the replicative processes involved in HIV-1 infection of selected human fetal neural cell populations in vitro. To determine whether infection of the human fetal dorsal root ganglia (DRG) glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase (RT) assays were performed. Direct assay of HIV-1 infected neural cell supernatants as well as exposure of permissive SupT1 cells to these HIV-1-infected neural cell supernatants detected no RT activity in either the HIV-1-infected DRG glial cell supernatants or the SupT1 cell supernatants. When SupT1 cells were cocultivated with the HIV-1-infected neural cells for 24-hr intervals, RT activity was detected in the SupT1 supernatants from cocultures initiated less than 2 days after infection (most likely resulting from infectious input virus) but not from cocultures initiated on 3, 5, 10, and 30 days after infection. Hybridization analysis demonstrated transient expression of HIV-1 cytoplasmic mRNA with accumulation reaching a maximum level by 2 to 3 days postinfection, declining thereafter with low, but detectable, levels at 16 days postinfection. In addition, polymerase chain reaction amplification in conjunction with DNA blot hybridization detected HIV-1-specific proviral DNA at 3 days postinfection. Cumulatively, these data suggest that HIV-1 infection of human fetal DRG glial cells culminates in a nonproductive infection with expression of at least a fraction of the virus genome but no detectable infectious virus production.

Cells, Cultured↗
PubMed1989

Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4A epitope.

Human immunodeficiency virus type 1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome-associated neurological dysfunction, and it is believed that the presence of CD4 in the nervous system may be involved in the susceptibility of selected neural cell populations to HIV-1 infection. We previously demonstrated (B. Wigdahl, R. A. Guyton, and P. S. Sarin, Virology 159:440-445, 1987) that glial cells derived from human fetal dorsal root ganglion (DRG) are susceptible to HIV-1 infection and subsequently express at least a fraction of the virus genome. In contrast to HIV-1 infection of CD4+ lymphocytes, which can be blocked by treatment with monoclonal antibodies directed against the HIV-1-binding region of CD4 (T4A epitope), treatment of human fetal DRG glial cells with similar antibodies resulted in only a slight reduction in HIV-1-specific gag antigen expression. In addition, preincubation of the HIV-1 inoculum prior to infection with HIV-1-neutralizing antiserum did not reduce HIV-1 gag antigen expression in these cells. Furthermore, we were unable to detect the synthesis or accumulation of the CD4 molecule in neural cell populations derived from DRG. However, a protected CD4-specific RNA fragment was detected in RNA isolated from human fetal DRG and spinal cord tissue by an RNase protection assay with a CD4-specific antisense RNA probe. RNA blot hybridization analysis of total cellular RNA isolated from human fetal DRG and spinal cord demonstrated specific hybridization to an RNA species that comigrated with the mature 3.0-kilobase CD4 mRNA as well as two unique CD4 RNA species with relative molecular sizes of approximately 5.3 and 6.7 kilobases. Furthermore, all three CD4-related RNA species were polyadenylated when isolated from human fetal spinal cord tissue. These data suggest that HIV-1 infection of human fetal DRG glial cells may proceed via a mechanism of viral entry independent of the T4A epitope of CD4.

Antibodies, Monoclonal↗
PubMed1989

CD4-independent infection of human neural cells by human immunodeficiency virus type 1.

A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.

Antibodies, Viral↗
PubMed1989

Transcriptional activity of the herpes simplex virus genome during establishment, maintenance, and reactivation of in vitro virus latency.

We previously have described a model of in vitro herpes simplex virus (HSV) latency in which latent infection was (i) established with human leukocyte interferon (IFN-alpha) in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir); (ii) maintained after termination of combined inhibitor treatment by incubation at 40.5 degrees, and (iii) reactivated by either reducing the incubation temperature to 37 degrees or by superinfecting at the elevated temperature with human cytomegalovirus (HCMV). We now report the use of this system to examine the transcriptional activity of the HSV genome during establishment, maintenance, and reactivation of HSV latency in vitro. Numerous species of virus-specific polyadenylated RNAs were present during the first 3 days of combined BVDU and IFN-alpha treatment of HSV type 1 (HSV-1)-infected human fetus lung fibroblast cells. However, after 7 days of combined inhibitor treatment, only a very small quantity of virus-specific RNA could be detected utilizing the short unique region of the HSV-1 genome as probe. After terminating combined BVDU and IFN-alpha treatment and increasing the temperature from 37 to 40.5 degrees on day 7 after infection, virus-specific RNA was undetectable by RNA blot hybridization analysis; however, a small amount of HSV-specific RNA was detected in 2% of the cells by in situ hybridization. The HSV-1 transcriptional products produced after HCMV superinfection in the presence of selected inhibitors of macromolecular synthesis also were examined and demonstrated that the efficient activation of HSV-1 immediate-early gene transcription required the expression of not only immediate-early HCMV gene product(s), but also at least a subset of early-late gene products.

Blotting, Northern↗
PubMed1987

Human immunodeficiency virus infection of the developing human nervous system.

Human immunodeficiency virus (HIV), the etiologic agent of acquired immune deficiency syndrome (AIDS) and AIDS-related complex, has recently been implicated as a factor in the development of AIDS-related neurologic dysfunction and may be responsible for an increasing number of neonatal immunologic and neurologic disorders. However, as yet there is no model system available to investigate the interaction of HIV with the developing human nervous system in vitro. To approximate the intracellular events associated with HIV infection of the human fetus nervous system we infected cells obtained by enzymatic dissociation of aborted human fetus dorsal root ganglia and their attached spinal roots and nerves. The expression of the HIV gag gene protein products (p17 and p24) was detected in a subpopulation of cells with a nonneuronal morphology, reaching a maximum within 3 days. Although 70% of the nonneuronal cells were p17- and p24-positive 3 days after infection, a majority of the cell population survived acute HIV infection, with the expression of p17 and p24 decreasing below the limit of detection by 12 days postinfection. This system may prove useful for examining the neuropathology and neurobiology of acute, persistent, or latent HIV infection of the developing human nervous system.

Antigens, Viral↗
PubMed1987

Activation of latent herpes simplex virus type 2 infection in vitro requires a (E)-5-(2-bromovinyl)-2'-deoxyuridine-sensitive gene function.

Previous studies have shown that herpes simplex virus (HSV) type 2 (HSV-2) can be maintained in a latent state in a limited number of cells by elevating the incubation temperature after treatment of HSV-infected human fetus lung fibroblast cells with metabolic inhibitors. Superinfection with human cytomegalovirus (HCMV) of latently infected cells maintained at the elevated temperature reactivated latent virus. In addition, superinfection with temperature-sensitive mutants indicated that reactivation of latent HSV in vitro did not require the expression of late gene function(s) of the superinfecting virus. We now report the (i) design of an in vitro HSV-2-latency system in which a higher percentage of cells contain a virus genome that can be activated; and (ii) subsequent use of this system to further characterize the virus activation process. Superinfection with a transcription-negative temperature-sensitive mutant of HSV type 1 (HSV-1) did not reactivate HSV-2-replication, suggesting that adsorption and penetration of the superinfecting virus were not sufficient for reactivation of the latent virus. Furthermore, superinfection with HSV-1 in the presence of (E)-5-(2-bromovinyl)-2'-deoxyuridine did not reactivate HSV-2 replication, suggesting that the expression of the immediate-early gene products are not sufficient for HSV-2 reactivation. Collectively, these data suggest that in addition to the expression of immediate-early gene function(s) at least a subset of early HSV-1 gene products are required for reactivation of latent HSV-2 in vitro.

Antiviral Agents↗
PubMed1986

Varicella-zoster virus infection of human sensory neurons.

Primary varicella-zoster virus (VZV) infection of humans may result in latent infection of sensory neurons in the peripheral nervous system. To examine the interaction of VZV with the sensory neuron we infected immunochemically defined human neurons with cell-associated VZV. Utilizing double-label immunofluorescence technology, a VZV-specific glycoprotein and a nonglycosylated phosphoprotein were detected in human fetus dorsal root ganglion (DRG) neurons, as defined by the presence of the neuron-specific enolase isoenzyme and the A2B5 ganglioside antigen, respectively. In addition to VZV antigen expression, progressive virus-induced cytopathic damage (neuronal enlargement and nuclear granulation of a fraction of the neuron population) was evident. As determined by transmission electron microscopy, VZV-infected human fetus DRG neurons contained empty and complete nucleocapsids with numerous pleomorphic virus particles in the cytoplasm, often in association with vacuoles. Although virus-specific antigen expression, particle synthesis, and cytopathic effects were observed in the human neuron population, neurons were less susceptible to VZV-induced cytopathic damage than supporting nonneuronal cells, suggesting neuronal modulation of VZV infection in vitro. This system provides the first model to examine the neuron- and virus-specific gene(s) and gene product(s) pertinent to the interaction of VZV with the human neuron.

Cells, Cultured↗