Biomedical subjects

Frank Kirchhoff

Publications and source records attributed to Frank Kirchhoff.

53 records · Page 3Linked to original sources

PubMed2004

Nef associates with p21-activated kinase 2 in a p21-GTPase-dependent dynamic activation complex within lipid rafts.

We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.

Cell Line↗

PubMed2004

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors.

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.

Amino Acid Substitution↗

PubMed2003

Alterations in HIV-1 LTR promoter activity during AIDS progression.

HIV-1 variants evolving in AIDS patients frequently show increased replicative capacity compared to those present during early asymptomatic infection. It is known that late stage HIV-1 variants often show an expanded coreceptor tropism and altered Nef function. In the present study we investigated whether enhanced HIV-1 LTR promoter activity might also evolve during disease progression. Our results demonstrate increased LTR promoter activity after AIDS progression in 3 of 12 HIV-1-infected individuals studied. Further analysis revealed that multiple alterations in the U3 core-enhancer and in the transactivation-response (TAR) region seem to be responsible for the enhanced functional activity. Our findings show that in a subset of HIV-1-infected individuals enhanced LTR transcription contributes to the increased replicative potential of late stage virus isolates and might accelerate disease progression.

Adolescent↗

PubMed2003

Mutations in the C3 region of human and simian immunodeficiency virus envelope have differential effects on viral infectivity, replication, and CD4-dependency.

Residues within the highly conserved C3 region of human and simian immunodeficiency virus (HIV, SIV) envelope proteins (Envs) bind directly to the cellular CD4 receptor. However, substitutions of D385, which is critical for CD4 engagement along with other changes such as G382R, G383R, frequently arise in SIV mac-infected macaques. We investigated the influence of substitutions in the SIVmac and HIV-1 C3 regions on viral entry, dependence on CD4, and replication. Mutations flanking the C3 region such as G382R or V388A enhanced and changes within the C3 region (i.e., G383R or D385N) impaired SIVmac infectivity. Several naturally occurring sequence variations in the SIVmac Env C3 region facilitated CD4-independent membrane fusion but abrogated viral replication, suggesting that efficient infection requires additional changes elsewhere in Env. Substitutions of S365R and D368G in the HIV-1 Env, which correspond to G382 and D385 in SIVmac Env, consistently impaired viral infectivity. In contrast, mutation of D368N resulted in a virus that could not spread in cells expressing low levels of CD4, but which replicated efficiently when high levels of CD4 were expressed. Thus, changes in the C3 region of HIV-1 or SIVmac Env can have differential effects on viral infectivity and CD4-dependency. We conclude that substitutions flanking the C3 region in SIVmac Env such as G382R or V388A represent one step toward adaptation to growth in target cells expressing low CD4 levels, whereas changes within the C3 region that disrupt CD4 binding might indicate the emergence of CD4-independent variants at later stages of infection, which could potentially broaden viral tropism.

Amino Acid Substitution↗

PubMed2003

Enhanced CD4 down-modulation by late stage HIV-1 nef alleles is associated with increased Env incorporation and viral replication.

Three viral proteins participate in the down-modulation of CD4 in human immunodeficiency virus type 1 (HIV-1)-infected cells. The underlying mechanisms have been extensively investigated. However, the physiological relevance of this phenomenon remains poorly understood. To address the role of CD4 down-modulation in HIV-1 pathogenesis in vivo, we have characterized the functional properties of nef alleles isolated from seven HIV-1-infected patients at either the stage of AIDS (late alleles) or during the asymptomatic phase of infection (early alleles). HIV-1 variants carrying these nef alleles showed striking differences in CD4 down-modulation, virus infectivity, and replication properties. Infection of T cells with late strains resulted in production of viral particles with enhanced infectivity, as compared with variants carrying early nef alleles. These differences in infectivity were observed only when viruses were produced in cells with high levels of the viral receptor, suggesting a functional link between CD4 levels and the ability of Nef to down-modulate CD4 and to enhance viral infectivity. Similarly, late nef alleles were substantially more active than early nef genes in stimulating HIV-1 replication in high CD4-positive cells, including primary lymphocytes, but not in cells expressing low levels of the CD4 receptor. Single-round assays showed that differences in infectivity between late and early strains are largely reduced when evaluated in target cells with high levels of CD4, suggesting that the inhibitory effect occurs at the entry step. Supporting this, enhanced CD4 down-modulation by late nef alleles was associated with higher levels of envelope incorporation into viral particles, a phenomenon that likely accounted for the augmented infectivity. Our data suggest a mechanistic link between the Nef-mediated CD4 down-modulation and the enhancement of replication in CD4-positive lymphocytes. As progression to disease occurs, HIV-1 Nef variants with enhanced ability to down-modulate CD4 are selected. These strains efficiently overcome the deleterious effects of CD4 and replicate more aggressively in CD4-positive primary lymphocytes. These results highlight the importance of the virus-induced CD4 down-modulation in HIV-1 pathogenesis.

Alleles↗

PubMed2003

Bone marrow-derived cells expressing green fluorescent protein under the control of the glial fibrillary acidic protein promoter do not differentiate into astrocytes in vitro and in vivo.

Differentiation of bone marrow (BM) cells into astroglia expressing the glial fibrillary acidic protein (GFAP) has been reported in vitro and after intracerebral or systemic BM transplantation. In contrast, recent data suggest that astrocytic differentiation does not occur from BM-derived cells in vivo. Using transgenic mice that express the enhanced green fluorescent protein (GFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter, we investigated the potential of adult murine BM-derived cells to differentiate into macroglia. In the brains of GFAP-GFP transgenic mice, astrocytes were brightly fluorescent from the expression of GFP. When BM from these animals was transplanted into lethally irradiated wild-type animals, the transgene was detected in the reconstituted hematopoietic system, but no GFP expression was found in the nervous system. In contrast, GFAP-GFP neuroectodermal anlage grafted into adult wild-type striatum gave rise to GFP-expressing astrocytes. Because cerebral ischemia has been suggested to promote the differentiation of BM-derived cells into astrocytes, BM chimeric mice were subjected to focal cerebral ischemia. No GFP-positive cells were found in the ischemic or contralateral hemispheres of these brains. Even after direct injection of GFAP-GFP transgenic BM cells into wild-type striatum, no GFP-expressing astroglia were detected. To test the hypothesis that the in vitro environment might be more permissible for astroglial differentiation, we cultured BM from mice that constitutively express GFP, BM cells expressing GFP from a retroviral vector, and BM from GFAP-GFP transgenic mice on astrocytes and on organotypic hippocampal slices. In all experimental paradigms, BM-derived cells were found to differentiate into ramified microglia but not into GFAP-expressing astrocytes.

Animals↗

PubMed2003

Neuronal or glial progeny: regional differences in radial glia fate.

The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS.

Animals↗

PubMed2003

Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus.

Recent data have suggested the existence of direct signaling pathways between glial cells and neurons. Here we report the coexistence of distinct types of cells expressing astrocyte-specific markers within the hippocampus that display diverse morphological, molecular, and functional profiles. Usage of transgenic mice with GFAP promoter-controlled enhanced green fluorescent protein (EGFP) expression allowed the identification of astroglial cells after fresh isolation or in brain slices. Combining patch-clamp recordings and single-cell reverse transcription-PCR, we distinguished two morphologically distinct types of EGFP-positive cells, one expressing glutamate transporters and the other expressing ionotropic glutamate receptors. None of the EGFP-positive cells coexpressed glutamate receptors and transporters. Subpopulations of glutamate receptor-bearing EGFP-positive cells expressed AN2, the mouse homolog of the rat NG2 proteoglycan or transcripts for excitatory amino acid carrier 1, a neuronal glutamate transporter. Our data demonstrate the presence of distinct, independent populations of cells with astroglial properties in the developing hippocampus that can differently modulate neuronal signaling pathways. The observed heterogeneity of cells with GFAP promoter-regulated EGFP expression and S100beta/GFAP immunoreactivity challenges the hitherto accepted definition of astrocytes.

Amino Acid Transport System X-AG↗

PubMed2003

Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical analysis.

Kainate-induced seizures increase hippocampal neurogenesis. Glial fibrillary acidic protein-positive astrocytes with radial processes in the dentate gyrus share many of the characteristics of radial glia and appear to act as precursor cells for adult dentate neurogenesis. Using the chemoconvulsant kainate and transgenic mice with human glial-fibrillary acidic protein (hGFAP) promoter-controlled enhanced green fluorescent protein (EGFP) expression, we examined the proliferation, morphology and electrophysiological properties of astrocytes in the neurogenic subgranular zone of the dentate gyrus in control animals and upon the induction of seizure-induced cell proliferation, three days post-kainate. EGFP-positive cells with and without radial processes could easily be distinguished. Kainate treatment caused a significant increase in the total number of proliferating EGFP-positive cells, particularly a tenfold elevation in the number of proliferating radial glia-like astrocytes, and also caused a preferential shift in the dividing cell population towards cells expressing EGFP. Immunohistochemical analysis revealed a surprisingly low proportion of cells coexpressing the astroglial marker S100beta and EGFP. Kainate increased the number of EGFP-positive, S100beta-positive and S100beta-positive-EGFP-positive astrocytes in the subgranular zone. We also report a subset of faintly EGFP-positive cells expressing markers of early neuronal differentiation. Patch-clamp analysis revealed the presence of three functionally different populations of EGFP-positive cells in both kainate and control tissue. We conclude that there is an early increase in proliferating radial glia-like astrocytes in the dentate after kainate-induced seizures, consistent with a recruitment of precursors for seizure-induced neurogenesis.

Animals↗

PubMed2003

Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles.

Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA 98:12144-12149, 2001). These Nef functions could contribute to impaired CD4(+)-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether nef alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1 nef alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV) nef alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably, nef genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas nef alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G protein-coupled receptor.

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

Animals↗

PubMed2002

The most frequent naturally occurring length polymorphism in the HIV-1 LTR has little effect on proviral transcription and viral replication.

About 38% of primary HIV-1 long terminal repeats (LTRs) contain an insertion (consensus: 5prime prime or minute-ACYGCTGA-3prime prime or minute), termed the most frequent naturally occurring length polymorphism (MFNLP). The MFNLP binds several transcription factors and might affect HIV-1 replication and disease progression in infected individuals. However, its relevance for proviral transcription and for HIV-1 replication in primary cells is unclear. We utilized HIV-1 NL4-3 LTR variants to investigate the effect of the MFNLP on 5prime prime or minuteLTR transcriptional activity in various cell types. Notably, viral promoter activity was studied in primary cells in the context of the integrated provirus, using both single cycle assays with pseudotyped Luciferase reporter viruses and replication-competent HIV-1 mutants. Our results demonstrate that the presence, absence, or duplication of the 5prime prime or minute-ACYGCTGA-3prime prime or minute motif has little effect on viral promoter activity in T cell lines, peripheral blood mononuclear cells (PBMC), and monocyte-derived macrophages (MDM). Furthermore, all HIV-1 LTR variants showed efficient induction upon stimulation with TPA and/or ionomycin and replicated with comparable efficiency in a human T cell line and in PBMC. Thus, the MFNLP does not significantly affect HIV-1 5prime prime or minuteLTR transcriptional activity and viral replication in primary cells, suggesting that this common sequence variation has little impact on the clinical course of HIV-1 infection.

Base Sequence↗

PubMed2002

Hemofiltrate CC chemokine 1[9-74] causes effective internalization of CCR5 and is a potent inhibitor of R5-tropic human immunodeficiency virus type 1 strains in primary T cells and macrophages.

Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.

Anti-HIV Agents↗

PubMed2002

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism.

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.

Amino Acid Sequence↗

PubMed2002

T-cell receptor:CD3 down-regulation is a selected in vivo function of simian immunodeficiency virus Nef but is not sufficient for effective viral replication in rhesus macaques.

We investigated the function of severely truncated simian immunodeficiency virus (SIV) Nef proteins (tNef) in vitro and in vivo. These variants emerged in rhesus monkeys infected with SIVmac239 containing a 152-bp deletion in the nef-unique region and have been suggested to enhance SIV virulence (E. T. Sawai, M. S. Hamza, M. Ye, K. E. Shaw, and P. A. Luciw, J. Virol. 74:2038-2045, 2000). We found that the tNef proteins were unable to down-regulate the cell surface expression of major histocompatibility complex class I proteins, CD4, and CD28 and neither stimulated SIV replication nor enhanced virion infectivity. The tNef proteins did efficiently down-regulate T-cell receptor (TCR):CD3 cell surface expression. Nevertheless, the SIVmac239 tnef variants were strongly attenuated in six infected juvenile rhesus macaques. Thus, while the ability of SIV Nef to down-modulate TCR:CD3 cell surface expression apparently confers a selective advantage in vivo, it is insufficient for efficient viral replication in infected macaques. Additional mutations elsewhere in SIVmac239 tnef genomes are required for a virulent phenotype.

Amino Acid Sequence↗

PubMed2001

Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices.

Pathologic impacts in the brain lead to a widespread activation of microglial cells far beyond the site of injury. Here, we demonstrate that glial Ca2+ waves can trigger responses in microglial cells. We elicited Ca2+ waves in corpus callosum glial cells by electrical stimulation or local adenosine triphosphate (ATP) ejection in acute brain slices. Macroglial cells, but not microglia, were bulk-loaded with Ca2+-sensitive dyes. Using a transgenic animal in which astrocytes were labeled by the enhanced green fluorescence protein (EGFP) allowed us to identify the reacting cell populations: the wave activated a Ca2+ response in both astrocytes and non-astrocytic glial cells and spread over hundreds of micrometers even into the adjacent cortical and ventricular cell layers. Regenerative ATP release and subsequent activation of metabotropic purinergic receptors caused the propagation of the glial Ca2+ wave: the wave was blocked by the purinergic receptor antagonist Reactive Blue 2 and was not affected by the gap junction blocker octanol, but enhanced in Ca2+ free saline. To test whether microglial cells respond to the wave, microglial cells were labeled with a dye-coupled lectin and membrane currents were recorded with the patch-clamp technique. When the wave passed by, a current with the characteristics of a purinergic response was activated. Thus, Ca2+ waves in situ are not restricted to astrocytic cells, but broadly activate different glial cell types.

Adenosine Triphosphate↗

PubMed1992

GABA Triggers a [Ca2+]i Increase in Murine Precursor Cells of the Oligodendrocyte Lineage.

The development of oligodendrocytes from their precursor cells can be studied in vitro by using a culture system in which cells can be identified at different developmental stages. We used this culture system to compare the effect of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) using fura-2 fluorescence systems. Application of GABA evoked transient [Ca2+]i increases in precursor cells. In contrast, [Ca2+]i levels were not affected in oligodendrocytes, which were identified by their positive labelling with the antibody O1. The precursor cells, identified by a lack of O1 staining, responded to GABA in the concentration range between 10-6 and 10-4 M. Since muscimol mimicked and bicuculline as well as picrotoxin blocked the GABA response, we conclude that the response is mediated by activation of GABAA receptors. The involvement of Ca2+ channels is inferred from the observation that the [Ca2+]i changes could be blocked by nifedipine or by omitting Ca2+ from the bath solution. Both GABAA receptors and Ca2+ channels have been previously identified on these precursor cells with the aid of the patch-clamp technique. We thus propose the following mechanism to explain our observations: the Cl- efflux via the GABA receptor depolarizes precursor cells, and this depolarization leads to activation of Ca2+ channels, resulting in an influx of Ca2+ and the observed rise in cytosolic [Ca2+]. Although its physiological importance is speculative, this event could serve as a signal from GABAergic neurons to glial precursor cells.

Journal Article↗