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C Kunsch

Publications and source records attributed to C Kunsch.

26 records · Page 2Linked to original sources
PubMed1992

Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation.

Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappa B DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappa B motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappa B complex. Similarly, in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappa B activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappa B motifs present.

Base Sequence↗
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↗
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↗