Biomedical subjects

Thomas N Kledal

Publications and source records attributed to Thomas N Kledal.

8 recordsLinked to original sources

PubMed2006

Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity.

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response element-driven transcriptional activity in a pertussis toxin-sensitive manner. Galpha(s) and Galpha(q) were not activated constitutively as determined by the lack of cAMP production, the lack of inositol phosphate turnover, and the lack of activities of the transcription factors: cAMP response element-binding protein and nuclear factor-kappaB. Immunohistochemistry and confocal microscopy of FLAG- and green fluorescent protein-tagged EBI2 revealed cell-surface expression. A putative N-terminal truncated version of EBI2, delta4-EBI2, showed similar expression and signaling through Galpha(i) as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based on the constitutive signaling and cellular expression pattern of EBI2, it is suggested that it may function in conjunction with BILF1 in the reprogramming of the cell during EBV infection.

Animals↗

PubMed2006

Targeting herpesvirus reliance of the chemokine system.

Viral infections depend on an intimate relationship between the infectious agent and the host cells. Viruses need the host cells for replication, while the innate- and adaptive- immunesystem of the host is fighting to kill the infected cell in order to clear out the pathogen and survive the infection. However, since both virus and host exist, the organisms struggle must reach an ecological equilibrium. Among the best-studied interactions between viruses and the host immune system are those between herpesviruses and their hosts. Herpesviruses are known to devote a significant part of their large genomes on immuno-modulatory genes, some encoding chemokines or chemokine receptors. These genes, which may be dispensable for viral replication in vitro, are highly important for viral growth in vivo, for viral dissemination and disease progression. Indeed, all beta and gamma-herpesviruses have acquired homologs of both chemokines and chemokine receptors belonging to the 7 transmembrane (7TM) spanning, G protein-coupled receptor family. 7TM receptors are very efficient drug targets and are currently the most popular class of investigational drug targets. A notable trait for the virus encoded chemokine receptors seems to be their constitutive activity. The biological function of the constitutive activity is still unclear, but it has become clear that the receptors are involved in important parts of the viral lifecycle in vivo, and that the receptor signaling is involved in gamma-herpesvirus mediated cell transformation. Therefore, blocking the signaling of these receptors will provide an efficient and highly specific way to inhibit viral replication in vivo and disease progression in the hosts.

Animals↗

PubMed2005

High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3.

The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded by equine herpesvirus 2 (EHV2), the DRY motif is substituted with a DTW motif. Nevertheless, this receptor signaled with high constitutive activity through Gi as determined by a receptor-mediated inhibition of forskolin-induced cAMP-production and by an induction of the serum response element-driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor of activated T cells. Coexpression of the ORF74-EHV2 receptor with the promiscuous G protein Gqi4myr supported the constitutive Gi activation as determined by inositol phosphate turnover and CREB activation. The constitutive activity was inhibited by nonpeptide inverse agonists with micromolar potencies, and the chemokine CXCL6 acted as a high-affinity agonist. It is noteworthy that reconstitution of the DRY motif resulted in a 4- to 5-fold decrease of the constitutive activity. Both the wild type and the receptor with the reconstituted DRY motif were expressed at the cell surface as indicated by immunohistochemistry and enzyme-linked immunosorbent assay analysis. It is concluded that the Arg of the DRY motif in transmembrane helix 3 is not essential for G protein coupling based on the constitutive as well as the ligand-mediated activity observed for ORF74-EHV2.

Amino Acid Motifs↗

PubMed2005

Epstein-Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor.

Both beta- and gammaherpesviruses encode G protein-coupled receptors (GPCRs) with unique pharmacological phenotypes and important biological functions. An example is ORF74, the gamma2-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR, which is highly constitutively active and considered the key oncogene in Kaposi's sarcoma pathogenesis. In contrast, the current annotation of the Epstein-Barr virus (EBV) genome does not reveal any GPCR homolog encoded by this human oncogenic gamma1-herpesvirus. However, by employing bioinformatics, we recognized that the previously established EBV open reading frame BILF1 indeed encodes a GPCR. Additionally, BILF1 is a member of a new family of related GPCRs exclusively encoded by gamma1-herpesviruses. Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelial cell line revealed that BILF1 is expressed as an approximately 50-kDa glycosylated protein. Immunocytochemistry and confocal microscopy revealed that BILF1 localizes predominantly to the plasma membrane, similar to the localization of KSHV ORF74. Using chimeric G proteins, we found that human and rhesus EBV-encoded BILF1 are highly potent constitutively active receptors, activating Galphai. Furthermore, BILF1 is able to inhibit forskolin-triggered CREB activation via stimulation of endogenous G proteins in a pertussis toxin-sensitive manner, verifying that BILF1 signals constitutively through Galphai. We suggest that EBV may use BILF1 to regulate Galphai-activated pathways during viral lytic replication, thereby affecting disease progression.

Amino Acid Sequence↗

PubMed2005

Predicting coding potential from genome sequence: application to betaherpesviruses infecting rats and mice.

Prediction of protein-coding regions and other features of primary DNA sequence have greatly contributed to experimental biology. Significant challenges remain in genome annotation methods, including the identification of small or overlapping genes and the assessment of mRNA splicing or unconventional translation signals in expression. We have employed a combined analysis of compositional biases and conservation together with frame-specific G+C representation to reevaluate and annotate the genome sequences of mouse and rat cytomegaloviruses. Our analysis predicts that there are at least 34 protein-coding regions in these genomes that were not apparent in earlier annotation efforts. These include 17 single-exon genes, three new exons of previously identified genes, a newly identified four-exon gene for a lectin-like protein (in rat cytomegalovirus), and 10 probable frameshift extensions of previously annotated genes. This expanded set of candidate genes provides an additional basis for investigation in cytomegalovirus biology and pathogenesis.

Amino Acid Sequence↗

PubMed2004

Cytomegalovirus infection impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine in transplant arteriosclerosis.

BACKGROUND: We hypothesized that cytomegalovirus (CMV) may contribute to the vasculopathy observed in cardiac allograft recipients by impairing the endothelial nitric oxide synthase pathway. We focused on asymmetric dimethylarginine (ADMA, the endogenous inhibitor of nitric oxide synthase) as a potential mediator of the adverse vascular effect of CMV. METHODS AND RESULTS: Heart transplant recipients manifested elevated plasma ADMA levels compared with healthy control subjects. Transplant patients with CMV DNA-positive leukocytes had higher plasma ADMA concentrations and more extensive transplant arteriopathy (TA). Human microvascular endothelial cells infected with the CMV isolates elaborated more ADMA. The increase in ADMA was temporally associated with a reduction in the activity of dimethylarginine dimethylaminohydrolase (DDAH, the enzyme that metabolizes ADMA). Infected cultures showed high levels of oxidative stress with enhanced endothelial production of superoxide anion. CONCLUSIONS: CMV infection in human heart transplant recipients is associated with higher ADMA elevation and more severe TA. CMV infection in endothelial cells increases oxidative stress, impairs DDAH activity, and increases ADMA elaboration. CMV infection may contribute to endothelial dysfunction and TA by dysregulation of the endothelial nitric oxide synthase pathway.

Amidohydrolases↗

PubMed2002

Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes.

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

Animals↗

PubMed2002

Murine cytomegalovirus (CMV) M33 and human CMV US28 receptors exhibit similar constitutive signaling activities.

Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-kappaB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-kappaB activation. Interestingly, UL33-the HCMV-encoded structural homologue of M33-was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-kappaB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.

Animals↗