Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of angiogenic proliferative skin lesions (KS), as well as of several rare B-cell lymphomas (PEL and MCD). KS is particularly prevalent in AIDS patients and has now become the most common of all tumors in Southern Africa. In previous studies, we have: (A) Mapped and characterized patterns of KSHV mRNA and protein expression in latent and TPA-induced lytic PEL cell lines by primer extension, northern blots, sequencing of isolated cDNA clones, and indirect I FA with specific antibodies;(B) Established a biological assay system for primary de novo infection by KSHV in contact-inhibited adult dermal microvascular endothelial cells (DMVEC) in culture, which produces a proliferative spindle cell conversion phenotype associated with stable maintenance of a LANA1- positive episomal latent state, together with low level lytic cycle reactivation, features that closely mimic those found in nodular KS lesions;(C) Carried out gene array and real-time RT-PCR analyses of KSHV-infected compared to uninfected DMVEC, to identify and confirm a set of 27 highly up or down-regulated endothelial cell (EC) mRNAs;(D) Detected the absence or shutoff of several key vascular EC proteins in KSHV-infected spindle cells by IFA, including PECAM1/CD31, VWF, VE-Cadherin, and VCAM1;and (D) Demonstrated by immunohistochemistry (IHC) that essentially all LAN A1-positive fascicular and perivascular spindle cells of nodular KS also lack CD31 and VWF, but are consistently PROX1-positive. Our new goals in this proposal are to further evaluate the altered protein expression patterns and EC functions of both KS and cultured KSHV-infected spindle cells;to define novel features of the viral transcription profiles in KSHV-infected spindle DMVECs, and to assess the relative contributions of three KSHV proteins vFLIP, vMIR1 and vMIR2 to the apparent conversion of functionally intact metabolic vascular EC into proliferating pre-angiogenic EC with lymphatic features. The three Specific Aims include: (I) Further IHC analysis of the patterns of expression of key altered EC proteins in KSHV-infected spindle and neovascular vessel wall EC in KS tissue, and evaluating the functional status of KSHV-infected spindle DMVEC, including potential loss of normal metabolic EC functions such as forming 3-D tubules or the ability to respond to inflammatory cytokines;(II) Mapping novel mRNA transcripts, splicing patterns and promoters of KSHV that are associated with maintenance of latency or early lytic cycle reactivation in DMVECs, including generation and analysis of cDNA libraries and use of a new generation KSHV viral and cellular gene chip array;(III) Using high efficiency ectopic expression of isolated viral genes, siRNA interference and BAC knockout insertion or deletion genetic approaches, to identify processes that may specifically target key DMVEC proteins for down-regulation by either the KSHV vFLIP anti-apoptotic and NFkB-activating latency protein, or at the post-translational level by the KSHV vMIR and vMIR2 membrane ubiquitin E3 ligases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA113239-05
Application #
8018146
Study Section
Subcommittee G - Education (NCI)
Project Start
Project End
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
5
Fiscal Year
2010
Total Cost
$297,999
Indirect Cost
Name
Johns Hopkins University
Department
Type
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Shamay, Meir; Liu, Jianyong; Li, Renfeng et al. (2012) A protein array screen for Kaposi's sarcoma-associated herpesvirus LANA interactors links LANA to TIP60, PP2A activity, and telomere shortening. J Virol 86:5179-91
Woodard, Crystal; Shamay, Meir; Liao, Gangling et al. (2012) Phosphorylation of the chromatin binding domain of KSHV LANA. PLoS Pathog 8:e1002972
Choi, Young Bong; Sandford, Gordon; Nicholas, John (2012) Human herpesvirus 8 interferon regulatory factor-mediated BH3-only protein inhibition via Bid BH3-B mimicry. PLoS Pathog 8:e1002748
Henson, Brandon W; Johnson, Nicole; Bera, Alakesh et al. (2011) Expression of the HSV-1 capsid protein VP19C in Escherichia coli: a single amino acid change overcomes an expression block of the full-length polypeptide. Protein Expr Purif 77:80-5
Alcendor, Donald J; Knobel, Susan; Desai, Prashant et al. (2011) KSHV regulation of fibulin-2 in Kaposi's sarcoma: implications for tumorigenesis. Am J Pathol 179:1443-54
Hobbs, Robert F; Baechler, S├ębastien; Fu, De-Xue et al. (2011) A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy. Med Phys 38:2892-903
Alcendor, Donald J; Knobel, Susan M; Desai, Prashant et al. (2010) KSHV downregulation of galectin-3 in Kaposi's sarcoma. Glycobiology 20:521-32
Shamay, Meir; Greenway, Melanie; Liao, Gangling et al. (2010) De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins. J Biol Chem 285:36377-86
Nicholas, John (2010) Human herpesvirus 8-encoded cytokines. Future Virol 5:197-206

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