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Biomedical subjects

Don Ganem

Publications and source records attributed to Don Ganem.

At least 19 recordsLinked to original sources
PubMed2007

Induction of chemokine production by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells.

Infection with Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is linked strongly to development of KS, an endothelial neoplasm also characterized by striking neoangiogenesis and infiltration with inflammatory cells. To elucidate the links between endothelial infection and inflammation, primary human umbilical vein endothelial cells (HUVECs) were examined for the production of chemokines following latent KSHV infection. Several chemokines that are produced in the ground state, including MCP-1, NAP 2 and RANTES, are upregulated significantly by KSHV infection. Moreover, the chemokine CXCL16, which is nearly absent in uninfected cells, is induced significantly following infection. This induction is attributable primarily to expression of vFLIP, a known inducer of NF-kappaB. CXCL16 induces the chemotaxis of activated T cells, whose products have been proposed to positively regulate KS tumour-cell survival and growth. Whilst CXCL16 has also been proposed as a direct endothelial chemoattractant and mitogen, neither proliferation nor chemotaxis of HUVECs was observed following CXCL16 exposure. These results suggest that CXCL16 induction by KSHV contributes to the inflammatory phenotype of KS, but plays little role in the recruitment of endothelial spindle cells.

Cells, Cultured↗
PubMed2006

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.

A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.

Adult↗
PubMed2006

Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.

Ribonuclease L (RNase L) is an important effector of the innate antiviral response. Mutations or variants that impair function of RNase L, particularly R462Q, have been proposed as susceptibility factors for prostate cancer. Given the role of this gene in viral defense, we sought to explore the possibility that a viral infection might contribute to prostate cancer in individuals harboring the R462Q variant. A viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses identified the presence of gammaretroviral sequences in cDNA samples from seven of 11 R462Q-homozygous (QQ) cases, and in one of eight heterozygous (RQ) and homozygous wild-type (RR) cases. An expanded survey of 86 tumors by specific RT-PCR detected the virus in eight of 20 QQ cases (40%), compared with only one sample (1.5%) among 66 RQ and RR cases. The full-length viral genome was cloned and sequenced independently from three positive QQ cases. The virus, named XMRV, is closely related to xenotropic murine leukemia viruses (MuLVs), but its sequence is clearly distinct from all known members of this group. Comparison of gag and pol sequences from different tumor isolates suggested infection with the same virus in all cases, yet sequence variation was consistent with the infections being independently acquired. Analysis of prostate tissues from XMRV-positive cases by in situ hybridization and immunohistochemistry showed that XMRV nucleic acid and protein can be detected in about 1% of stromal cells, predominantly fibroblasts and hematopoietic elements in regions adjacent to the carcinoma. These data provide to our knowledge the first demonstration that xenotropic MuLV-related viruses can produce an authentic human infection, and strongly implicate RNase L activity in the prevention or clearance of infection in vivo. These findings also raise questions about the possible relationship between exogenous infection and cancer development in genetically susceptible individuals.

Adenocarcinoma↗
PubMed2006

A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses.

We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.

Algorithms↗
PubMed2006

Activation of NF-kappaB by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype.

Kaposi's sarcoma (KS) is an inflammatory angioproliferative lesion induced by the infection of endothelial cells with the KS-associated herpesvirus (KSHV). Infected endothelial cells assume an elongated (spindle) shape that is one of the histologic signatures of KS. In vitro, latent viral infection of primary endothelial cells (but no other cell type) strikingly recapitulates these morphological findings. Here we report that the spindling phenotype involves major rearrangement of the actin cytoskeleton and can be attributed to the expression of a single viral protein, vFLIP, a known activator of NF-kappaB. Consistent with this, the inhibition of NF-kappaB activation blocks vFLIP-induced spindling in cultured endothelial cells. vFLIP expression in spindle cells also induces the production of a variety of proinflammatory cytokines and cell surface adhesion proteins that likely contribute to the inflammatory component of KS lesions.

Actins↗
PubMed2006

Phosphorylation and function of the kaposin B direct repeats of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a protein, kaposin B, which is composed of multiple copies of 23-amino-acid direct repeats, termed DR2 and DR1. Kaposin B enhances the release of pathogenetically important proinflammatory cytokines by activating the p38 mitogen-activated protein kinase (MAPK)-MK2 kinase pathway and blocking cytokine mRNA decay. Here, we show that this mRNA stabilization function requires both the DR2 and DR1 elements of kaposin B; a monomeric form of the protein consisting of one copy of each repeat retains function. Furthermore, we show that p38 MAPK is capable of directly phosphorylating kaposin B in vitro and map the site of phosphorylation to a specific serine residue in DR1. Mutational ablation of this serine abolishes phosphorylation of the protein by p38 MAPK but does not affect kaposin B's ability to extend mRNA half-life.

Binding Sites↗
PubMed2006

Exploring the DNA binding interactions of the Kaposi's sarcoma-associated herpesvirus lytic switch protein by selective amplification of bound sequences in vitro.

The lytic switch protein RTA of Kaposi's sarcoma-associated herpesvirus (KSHV) can be targeted to DNA by either direct sequence-specific recognition or via protein-protein interactions with host transcription factors. We have searched for sequences capable of direct RTA binding by screening synthetic oligonucleotide pools and KSHV genomic libraries for RTA-interacting elements, using repeated cycles of in vitro binding followed by amplification of the bound sequences. Multiple low-affinity sequences were recovered from the random pools, with generation of only a weak consensus sequence. The genomic library, by contrast, yielded many biologically relevant fragments, most of which could be shown to interact with RTA in vitro and some of which likely play important regulatory roles in vivo. Surprisingly, the most highly selected fragment came from the promoter of a late gene (gB) and contained at least two direct RTA binding sites, as well as one RBP-Jkappa binding site. This raises the possibility that some late KSHV genes may also be subject to direct RTA regulation, though indirect models are not excluded.

Base Sequence↗
PubMed2005

MicroRNAs and viral infection.

MicroRNAs (miRNAs) play a pivotal role in the regulation of genes involved in diverse processes such as development, differentiation, and cellular growth control. Recently, many viral-encoded miRNAs have been discovered, for the most part in viruses transcribed from double-stranded DNA genomes. As with their cellular counterparts, the functions of most viral-derived miRNAs are unknown; however, functions have been documented or proposed for viral miRNAs from three different viral families-herpesviruses, polyomaviruses, and retroviruses. Several virus-encoded miRNAs have unique aspects to their biogenesis, such as the polymerase that transcribes them or their location within the precursor transcript. Additionally, viral interactions with cellular miRNAs have also been identified, and these have substantially expanded our appreciation of miRNA functions.

Animals↗
PubMed2005

SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells.

MicroRNAs (miRNAs) are small (approximately 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

Animals↗
PubMed2005

The kaposin B protein of KSHV activates the p38/MK2 pathway and stabilizes cytokine mRNAs.

Cytokine production plays a critical role in diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Here we show that a latent KSHV gene product, kaposin B, increases the expression of cytokines by blocking the degradation of their messenger RNAs (mRNAs). Cytokine transcripts are normally unstable because they contain AU-rich elements (AREs) in their 3' noncoding regions that target them for degradation. Kaposin B reverses this instability by binding to and activating the kinase MK2, a target of the p38 mitogen-activated protein kinase signaling pathway and a known inhibitor of ARE-mRNA decay. These findings define an important mechanism linking latent KSHV infection to cytokine production, and also illustrate a distinctive mode by which viruses can selectively modulate mRNA turnover.

Cell Line↗
PubMed2005

A virus-encoded inhibitor that blocks RNA interference in mammalian cells.

Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.

Cell Line↗
PubMed2005

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable.

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.

Cell Line↗
PubMed2005

RNAs in the virion of Kaposi's sarcoma-associated herpesvirus.

De novo infection of cultured cells with Kaposi's sarcoma-associated herpesvirus (KSHV) typically results in a latent infection. Recently, however, it has been reported that a subset of lytic mRNAs can be detected in cells shortly after KSHV infection; this expression is transient and eventually subsides, leading to latent infection (H. H. Krishnan et al., J. Virol 78:3601-3620, 2004). Since it has been shown that viral RNAs can be packaged into other herpesvirus virions, we sought to determine if KSHV virions contained RNAs and, if so, whether these RNAs contributed to the pool of lytic transcripts detected immediately after infection. Using DNA microarray, reverse transcription (RT)-PCR, and Northern blotting analyses, we identified 11 virally encoded RNAs in KSHV virions. These corresponded in size to the full-length mRNAs found in cytoplasmic RNA, and at least one was directly demonstrated to be translated upon infection in the presence of actinomycin D. Ten of these RNAs correspond to transcripts reported by Krishnan et al. at early times of infection, representing ca. 30% of such RNAs. Thus, import of RNAs in virions can account for some but not all of the early-appearing lytic transcripts. Quantitative RT-PCR analysis of infected-cell RNA demonstrated that most of the virion RNAs were very abundant at late times of infection, consistent with nonspecific incorporation during budding. However, the intracellular levels of one virion mRNA, encoding the viral protease, were much lower than those of transcripts not packaged in the virus particle, strongly suggesting that it may be incorporated by a specific mechanism.

Herpesvirus 8, Human↗
PubMed2005

Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus.

Infection of cultured cells with Kaposi's sarcoma associated herpesvirus (KSHV) typically establishes a latent infection, in which only a few viral genes are expressed. Recently, it has been reported that a subset of lytic genes are transiently expressed very early after viral entry but that this burst of abortive lytic gene expression is terminated with the supervention of latency (H. H. Krishnan, P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B. Chandran, J. Virol. 78:3601-3620, 2004). To identify molecules imported into cells by KSHV that might influence this gene expression program, we have examined the protein composition of the KSHV particle. Immunoblotting of virus particles demonstrated that RTA, the lytic switch protein, and RAP, a viral protein that is a transcriptional and cell cycle modulator, were both incorporated into virus particles. In a second approach, polypeptides isolated from purified virions were identified by mass-spectrometric analysis of their constituent tryptic peptides. With this approach we were able to identify 18 major virion proteins, including structural, regulatory, and signaling proteins of both viral and cellular origin.

Amino Acid Sequence↗
PubMed2005

Regulation of CD1d expression and function by a herpesvirus infection.

Little is known about the role of CD1d-restricted T cells in antiviral immune responses. Here we show that the lytic replication cycle of the Kaposi sarcoma-associated herpesvirus (KSHV) promotes downregulation of cell-surface CD1d. This is caused by expression of the 2 modulator of immune recognition (MIR) proteins of the virus, each of which promotes the loss of surface CD1d expression following transfection into uninfected cells. Inhibition of CD1d surface expression is due to ubiquitination of the CD1d alpha-chain on a unique lysine residue in its cytoplasmic tail, which triggers endocytosis. Unlike MIR-mediated MHC class I downregulation, however, CD1d downregulation does not appear to include accelerated lysosomal degradation. MIR2-induced downregulation of CD1d results in reduced activation of CD1d-restricted T cells in vitro. KSHV modulation of CD1d expression represents a strategy for viral evasion of innate host immune responses and implicates CD1d-restricted T cells as regulators of this viral infection.

Antigens, CD1↗
PubMed2004

Highly selective escape from KSHV-mediated host mRNA shutoff and its implications for viral pathogenesis.

During Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) lytic infection, many virus-encoded signaling molecules (e.g., viral G protein-coupled receptor [vGPCR]) are produced that can induce host gene expression in transiently transfected cells, and roles for such induced host genes have been posited in KS pathogenesis. However, we have recently found that host gene expression is strongly inhibited by 10-12 h after lytic reactivation of KSHV, raising the question of whether and to what extent de novo host gene expression induced by viral signaling molecules can proceed during the lytic cycle. Here, we show by microarray analysis that expression of most vGPCR target genes is drastically curtailed by this host shutoff. However, rare cellular genes can escape the host shutoff and are potently up-regulated during lytic KSHV growth. Prominent among these is human interleukin-6, whose striking induction may contribute to the overexpression of this cytokine in several disease states linked to KSHV infection.

DNA-Binding Proteins↗
PubMed2004

Lytic KSHV infection inhibits host gene expression by accelerating global mRNA turnover.

The stimulation of host gene expression by lytic gene products of Kaposi's sarcoma-associated herpesvirus (KSHV) has been proposed to play a critical role in KS development. We show, however, that lytic KSHV infection strongly inhibits host gene expression early in infection by accelerating global mRNA turnover. This function is mediated by KSHV ORF37, a homolog of a DNA exonuclease widely present in other herpesviruses but which in KSHV has uniquely evolved additional functions that mediate its participation in RNA degradation. The ability of KSHV to inhibit host gene expression has important implications for models of KS pathogenesis that invoke activation of host transcription in lytically infected cells as a source of angiogenic or oncogenic factors.

Amino Acid Motifs↗