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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Virus-Cell Interactions II Abstract 62 TARGETING EXTRACELLULAR HSP90 REDUCES KSHV GENE EXPRESSION DURING DE NOVO INFECTION BY INHIBITING MAPK PATHWAY ACTIVATION Zhiqiang Qin 1 , Jennifer Isaacs 2 , and Chris Parsons 1,3 Departments of 1 Medicine, 2 Pharmacology and 3 Microbiology/Immunology Hollings Cancer Center, Medical University of South Carolina Charleston, SC, USA Abstract Heat shock protein 90 (Hsp90) acts as a molecular chaperone orchestrating the folding of intracellular signaling proteins. Although extracellular (EC) domains for Hsp90 have been identified, the functional consequences of engaging EC Hsp90 are largely unknown. Signal transduction induced during KSHV receptor binding and entry is critical for de novo viral gene expression. Using PCR and immunofluorescence assays, we found that treatment of HeLa cells with a non-permeable compound targeting EC Hsp90, DNo, inhibited de novo expression of latent KSHV transcripts and LANA. Pre-treatment of cells with DNo did not reduce intracellular KSHV DNA content, and DNo maintained an inhibitory effect when treatment was initiated following viral incubation. In addition, no effect was observed following KSHV pre-incubation with two soluble Hsp90 protein isoforms or a mAb targeting a different EC Hsp90 domain. These results suggest that DNo interferes with post-entry events important for viral gene expression. Immunoblot analyses of signal transduction intermediates indicated that mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation were selectively inhibited in a dose- and time-dependent manner by DNo. Furthermore, DNo blocked KSHV-mediated induction of MEK and ERK phosphorylation corresponding to a reduction in LANA expression. Finally, constructs conferring constitutive expression of P-MEK and P-ERK, but not an ERK dominant negative construct, rescued KSHV gene expression in DNo-treated cells. These results suggest that EC Hsp90 plays an important role in KSHV-initiated signal transduction events and viral gene expression and that strategies targeting EC Hsp90 may have therapeutic benefit for KSHV-related disease. Presenting author Email: parsonch@musc.edu 90
The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Virus-Cell Interactions II Abstract 63 FAK AND SHP2 ARE REQUIRED FOR KSHV VGPCR SIGNALING Thomas Bakken*, Chris Boshoff**, Mark Cannon* *CIDMTR, Department of Medicine, University of Minnesota, ** C.R.U.K. Viral Oncology Group, University College London Abstract The KSHV vGPCR is a constitutively active homologue of CXCR1 and CXCR2. It shows potential both in vitro and in vivo to contribute to the proliferative, angiogenic and inflammatory components of KS biology. In transgenic mice, vGPCR causes KS-like tumors and potentiates the tumorgenicity of other viral proteins. These effects are likely a combination of direct vGPCR signaling and paracrine effects via vGPCR-induced elaboration of various growth and angiogenic factors. Inhibition of vGPCR function is a promising anti-KSHV strategy. We focus on identifying host cell pathways that are disrupted by vGPCR and could be targeted to interfere with vGPCR function. The protein phosphatase, Shp2, and the focal adhesion-associated kinase (FAK) are signal integration proteins with adaptor and enzymatic functions. Up-regulation of both is associated with various malignancies. Furthermore, both are the target of newly developed pharmacologic inhibitors (FAK inhibition in particular has shown promise in a mouse ovarian cancer model). Using shRNA knock-down and pharmacologic inhibition in HEK293, we find that vGPCR activation of MEK requires both FAK and Shp2. vGPCR-induced NFκB and AP-1 activation are also FAK-dependent, although only the former requires Shp2. These studies are being taken into primary endothelial cells in which the role of Shp2 and FAK in vGPCRmediated signaling and phenotypic changes will be assessed. Interfering with pathways that are upregulated by vGPCR is a promising anti-KSHV approach. We have identified two signaling integration proteins that are receiving attention in the medicinal chemistry field and that are required for important vGPCRmediated events. Presenting author Email: canno101@umn.edu 91
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