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.
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