Biomedical subjects

Don Ganem

Publications and source records attributed to Don Ganem.

32 records · Page 2Linked to original sources

PubMed2004

RBP-J (CSL) is essential for activation of the K14/vGPCR promoter of Kaposi's sarcoma-associated herpesvirus by the lytic switch protein RTA.

The Kaposi's sarcoma-associated herpesvirus (KSHV) gene product virally encoded G protein-coupled receptor (vGPCR) is a homolog of cellular GPCRs and has been proposed to play important roles in KSHV-induced angiogenesis. The most abundant vGPCR-containing transcripts are K14/vGPCR bicistronic RNAs that are strongly induced during lytic reactivation. Here we show that the promoter governing this transcript is strongly responsive to activation by the viral lytic switch protein RTA. By deletion mapping and scanning mutation analyses, we have identified three putative RTA response elements (A, B, and C) in this promoter. However, none of these sites appear to directly bind RTA in electrophoretic mobility shift assays (EMSA). Site C corresponds to a canonical binding site for RBP-J, a sequence-specific transcriptional repressor that is normally the target of Notch signaling. RBP-J can bind RTA and recruit it to its cognate recognition site; when this happens, the activation function of RTA can relieve RBP-J-mediated repression and upregulate expression of the targeted gene. EMSA studies reveal that both sites A and C can bind to RBP-J; sequence inspection reveals that site A is a novel functional variant of known RBP-J recognition sites. (Site B corresponds to an as-yet-unknown host DNA-binding protein.) The importance of sites A and C in vivo is underscored by the observation that K14/vGPCR promoter function is dramatically inhibited in cells genetically deficient in RBP-J. The regulation of K14/vGPCR transcripts by RBP-J raises the possibility that other modulators of Notch signaling might be able to induce expression of this RNA outside the context of lytic KSHV replication.

Base Sequence↗

PubMed2004

Inefficient establishment of KSHV latency suggests an additional role for continued lytic replication in Kaposi sarcoma pathogenesis.

Kaposi sarcoma-associated (KS-associated) herpesvirus (KSHV) infection is linked to the development of both KS and several lymphoproliferative diseases. In all cases, the resulting tumor cells predominantly display latent viral infection. KS tumorigenesis requires ongoing lytic viral replication as well, however, for reasons that are unclear but have been suggested to involve the production of angiogenic or mitogenic factors by lytically infected cells. Here we demonstrate that proliferating cells infected with KSHV in vitro display a marked propensity to segregate latent viral genomes, with only a variable but small subpopulation being capable of stable episome maintenance. Stable maintenance is not due to the enhanced production of viral or host trans-acting factors, but is associated with cis-acting, epigenetic changes in the viral chromosome. These results indicate that acquisition of stable KSHV latency is a multistep process that proceeds with varying degrees of efficiency in different cell types. They also suggest an additional role for lytic replication in sustaining KS tumorigenesis: namely, the recruitment of new cells to latency to replace those that have segregated the viral episome.

Burkitt Lymphoma↗

PubMed2003

Viral discovery and sequence recovery using DNA microarrays.

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

Base Sequence↗

PubMed2003

Lytic but not latent infection by Kaposi's sarcoma-associated herpesvirus requires host CSL protein, the mediator of Notch signaling.

Infection by Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a key factor in the development of KS. Both latent and lytic KSHV infection is observed in KS tumor cells, and both genetic programs contribute importantly to KS pathogenesis. The viral replication and transcription activator (RTA) protein is a transcription factor that controls the switch from latency to lytic replication. We have previously shown that RTA can activate the expression of several lytic viral genes in transfected cells by interaction with recombination signal sequence-binding protein-J kappa (RBP-J kappa, also called CSL), which in uninfected cells is a transcriptional repressor that is the target of the Notch-signaling pathway. The recognition that many KSHV lytic genes, including RTA itself, contain RBP-J kappa-binding sites raised the possibility that RBP-J kappa-mediated repression may be central to the establishment of latency. Here, we have tested this hypothesis by examining KSHV infection of RBP-J kappa-null murine fibroblasts. Our results show that KSHV latency is efficiently induced in such cells; however, the reactivation of lytic gene expression, viral DNA replication, and the release of progeny viruses are dramatically inhibited in the absence of RBP-J kappa. RBP-J kappa-mediated repression is therefore not essential for establishment of latent infection, but the RTA-mediated redirection of RBP-J kappa activity from repression to activation is critical for lytic viral replication.

Animals↗

PubMed2003

PHD domains and E3 ubiquitin ligases: viruses make the connection.

PHD domains constitute a widely distributed subfamily of zinc fingers whose biochemical functions have been unclear until now. Recently, several PHD-containing viral proteins have been identified that promote immune evasion by downregulating proteins that govern immune recognition. Studies show that these viral regulators lead to ubiquitination of their targets by functioning as E3 ubiquitin ligases -- an activity that requires the PHD motif. These are the first examples linking the PHD domain to E3 activity, but the recent discovery of PHD-dependent E3 activity in the cellular kinase MEKK1 and the close structural relation of PHD domains to RING fingers hint that many other PHD proteins might share this activity.

Amino Acid Sequence↗

PubMed2003

Host range of Kaposi's sarcoma-associated herpesvirus in cultured cells.

Difficulties in efficiently propagating Kaposi's sarcoma-associated herpesvirus (KSHV) in culture have generated the impression that the virus displays a narrow host range. Here we show that, contrary to expectation, KSHV can establish latent infection in many adherent cell lines, including human and nonhuman cells of epithelial, endothelial, and mesenchymal origin. (Paradoxically, the only lines in which we have not observed successful latent infection are cultured lymphoma cell lines.) In most latently infected lines, spontaneous lytic replication is rare and (with only two exceptions) is not efficiently induced by phorbol ester treatment-a result that explains the failure of most earlier studies to observe efficient serial transfer of infection. However, ectopic expression of the KSHV lytic switch protein RTA from an adenoviral vector leads to the prompt induction of lytic replication in all latently infected lines, with the production of infectious KSHV virions. These results indicate (i) that the host cell receptor(s) and entry machinery for KSHV are widely distributed on cultured adherent cells, (ii) that latency is the default pathway of infection, and (iii) that blocks to lytic induction are frequent and largely reside at or upstream of the expression of KSHV RTA.

Adenoviridae↗

PubMed2003

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus permits replication of terminal repeat-containing plasmids.

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus can associate with mitotic chromosomes and promote latent episome maintenance and segregation. Here we report that LANA also mediates the replication of plasmid DNAs bearing viral terminal repeats. The predicted secondary structure of LANA's C terminus reveals striking similarity to the known structure of the DNA-binding domain of Epstein-Barr virus EBNA1, despite the absence of primary sequence homology between these proteins, suggesting conservation of the key mechanistic features of latent gammaherpesvirus DNA replication.

Amino Acid Sequence↗

PubMed2002

Microarray-based detection and genotyping of viral pathogens.

The detection of viral pathogens is of critical importance in biology, medicine, and agriculture. Unfortunately, existing techniques to screen for a broad spectrum of viruses suffer from severe limitations. To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have developed a genomic strategy for highly parallel viral screening. The cornerstone of this approach is a long oligonucleotide (70-mer) DNA microarray capable of simultaneously detecting hundreds of viruses. Using virally infected cell cultures, we were able to efficiently detect and identify many diverse viruses. Related viral serotypes could be distinguished by the unique pattern of hybridization generated by each virus. Furthermore, by selecting microarray elements derived from highly conserved regions within viral families, individual viruses that were not explicitly represented on the microarray were still detected, raising the possibility that this approach could be used for virus discovery. Finally, by using a random PCR amplification strategy in conjunction with the microarray, we were able to detect multiple viruses in human respiratory specimens without the use of sequence-specific or degenerate primers. This method is versatile and greatly expands the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes.

DNA Viruses↗

PubMed2002

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway.

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. Here we show that KSHV RTA interacts with the RBP-Jkappa protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-Jkappa recognition sites on DNA and results in the replacement of RBP-Jkappa's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-Jkappa(-/-) mice, a defect that can be reversed by expression of RBP-Jkappa. In vitro, RTA binds to two adjacent regions of RBP-Jkappa, one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency.

Animals↗

PubMed2002

Modulation of host gene expression by the constitutively active G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpes virus (KSHV) infects B cells and microvascular endothelium,and is linked to both lymphoid and endothelial neoplasms. KSHV encodes a G protein-coupled receptor (v-GPCR) that can bind several CC and CXC chemokines but is able to signal in the absence of known ligands. This signaling can transform cultured fibroblasts, promote angiogenesis in vitro and in vivo, and activate the mitogen-activated protein kinase, c-Jun-NH(2)-terminal kinase, and p38 pathways. To assess the potential impact of v-GPCR signaling on host cell biology we have examined cellular gene expression in v-GPCR-transfected cells using DNA microarrays. v-GPCR expression up-regulated numerous cellular transcripts in both BJAB B cells and SLK endothelial cells, but with a remarkable degree of cell-type specificity. Among the most highly regulated genes in endothelial cells were the cytokines interleukin 6 and GRO alpha; several genes affecting endothelial/vascular growth and remodeling were also induced, including plasminogen, thrombomodulin, the urokinase-type plasminogen activator receptor, and to a modest extent vascular endothelial growth factor C. By contrast, the most highly regulated genes in B cells were the CC chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 1 beta. No genes other than members of the dual-specificity phosphatase family were induced in both cell lines. The results indicate that the effects of KSHV GPCR expression in these two target cell types differ considerably and suggest that signaling by this molecule may make different contributions to the pathogenesis of KSHV-related endothelial and lymphoproliferative lesions.

B-Lymphocytes↗

PubMed2002

Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection.

NKT cells are specialized cells of the immune system that bear both T cell and NK cell markers. Classical NKT cells display TCRs of restricted heterogeneity (Valpha14-Jalpha281) and recognize lipid antigens (e.g., alpha-galactosyl ceramide) presented by nonpolymorphic CD1 molecules. Recently, other nonclassical NKT subsets have been recognized, including NKT cells not reactive with CD1d-alpha-galactosyl ceramide complexes. The biological functions of these cells are unknown. Here, we show that nonclassical NKT cells that are CD1d restricted but nonreactive to alpha-GalCer are activated in response to hepatocytes expressing hepatitis B viral antigens in a transgenic mouse model of acute hepatitis B virus infection. Our results document the first in vivo function for such nonclassical NKT cells and suggest a role for these cells in the host response to HBV infection.

Acute Disease↗

PubMed2002

De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells.

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.

Caenorhabditis elegans Proteins↗

PubMed2001

Functional organization of MIR2, a novel viral regulator of selective endocytosis.

Kaposi's sarcoma-associated herpesvirus encodes two related proteins, MIR1 and MIR2, that lead to reduction of the cell surface levels of major histocompatibility complex class I and other polypeptides involved in immune recognition. MIR1 and MIR2 do not affect the assembly or transport of their target proteins through the secretory pathway; rather, they act to enhance the selective endocytosis of target chains from the cell surface. Sequence inspection reveals that the modulator of immune recognition (MIR) proteins contain an NH(2)-terminal zinc finger of the plant homeodomain (PHD) subfamily, two transmembrane (TM) domains, and a C-terminal conserved region (CR). Here we examine the transmembrane topology and functional organization of MIR2. Both the PHD domain and the CR are disposed cytosolically and are essential for MIR-mediated endocytosis. MIR proteins form homo-oligomers; this activity is independent of the PHD and CR elements and maps instead to the TM regions. Analysis of chimeras between MIR1 and MIR2 reveals that the TM regions also mediate target selectivity. Mutations that ablate the PHD or CR regions generate dominant negative phenotypes for major histocompatibility complex class I endocytosis. These findings suggest a domain organization for the MIR proteins, with the TM regions involved in target selection and the cytosolic PHD and CR domains involved in the possible recruitment of cellular machinery that directly or indirectly regulates internalization of target molecules.

Amino Acid Sequence↗

PubMed2000

On the control of late gene expression in Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8).

Herpesvirus late genes require viral DNA replication for maximal expression. Although late gene expression appears to require DNA replication in cis in alphaherpesviruses, studies in Epstein-Barr virus (EBV) suggest that this cis-requirement might not pertain to the gammaherpesviruses. Based on these findings, a system was created to investigate the elements required for the regulation of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8) late gene expression. The transcript of a classic late gene encoding the viral assembly protein was characterized and reporter genes driven by the assembly protein promoter region were constructed. Unlike the EBV case, expression of a reporter gene under the control of the assembly protein promoter did not display authentic regulation when removed from the context of the viral genome. Although reporter expression rose in cells displaying lytic replication, this expression was not diminished by specific inhibitors of viral DNA synthesis. Minimal core promoters were similarly unable to reproduce late gene regulation. These results suggest that proper KSHV late gene expression is likely to be dependent upon virus lytic replication in cis and indicate that the regulation of KSHV late genes more closely resembles that observed in herpes simplex virus than that described for EBV.

Amino Acid Sequence↗