Kaposi's sarcoma-associated herpesvirus (KSHV) is the apparent etiological cause of Kaposi's sarcoma, a neoplasm of frequent occurrence in HIV infected individuals. The KSHV genome is present in the spindle cells of virtually all KS lesions of both HIV positive and negative cases, and there is an excellent correlation between seroconversion to KSHV and development of KS. This herpesvirus is also strongly linked to two specific lymphoproliferative disorders, Primary Effusion Lymphoma (PEL) and Castleman's disease (CD). Less certain is the suggested role of KSHV in multiple myeloma. The mode of KSHV transmission is unknown and the virus may not be ubiquitous. The viral genome, now completely sequenced, encodes homologues of cellular cytokines and growth factors. Since standard herpesvirus drug treatments (e.g.., acyclovir) are not effective against KSHV, there is heightened interest in developing new KSHV antivirals. One promising new antiviral target is the DNA polymerase (Pol-8) and Processivity Factor (PF-8) complex of KCHV. PFs associate with their cognate DNA Pols, enabling them to synthesize extended stretches of DNA without dissociating from template. The PF genes of certain other herpesviruses are known to be required for viral DNA synthesis and infection. Indeed, only a few dNTPs are incorporated into DNA by Pol-8 alone in vitro, but when combined with PF-8, thousands of dNTPs are incorporated. Moreover, Pol-8 complexes and functions with only PF-8 and not PFs of other herpesviruses. The specificity of the PF-8/Pol-8 interaction, which is necessary for DNA synthesis, predicts that it should be possible to identify antivirals which are capable of blocking KSHV infection without perturbing normal cellular activities. The goal is to elucidate the mechanism of PF-8/Pol-8 processive DNA synthesis and to develop and employ a novel high throughput screening method that can be used to identify inhibitors that specifically block DNA synthesis and KSHV infection by targeting PF-8/Pol-8.
The aims are to determine the affinities, composition, and structures of PF-8 and Pol-8 both off and on DNA using analytical ultracetrifugation, surface plasmon resonance, mutagenesis, and crystallography. A high throughput assay to identify functional inhibitors of PF-8/Pol-8 will be developed and used to screen compounds from the NCI repository; blocking peptides with designs based on phage display and mutational analysis will also be tested. All of the inhibitors that block PF-8/Pol-8 DNA synthesis in vitro will be tested for their abilities to block KSHV infection.
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