PF-8 AND POL-8 OF KSHV AS TARGETS OF VIRAL DNA SYNTHESIS

描述：（改编自申请人的摘要）卡波西肉瘤相关 疱疹病毒（KSHV）是卡波西肉瘤的明显病因， 在HIV感染者中经常发生的肿瘤。KSHV基因组是 存在于几乎所有KS病变的梭形细胞中， 和阴性病例之间有很好的相关性 KSHV的血清转化和KS的发展。这种疱疹病毒也强烈 与两种特定的淋巴增生性疾病，原发性渗出性淋巴瘤 (PEL)Castleman病（CD）不太确定的是KSHV的作用 多发性骨髓瘤KSHV的传播方式尚不清楚，病毒可能 而不是无处不在。病毒基因组，现在完全测序，编码 细胞因子和生长因子的同系物。因为标准疱疹病毒 药物治疗（例如，阿昔洛韦）对KSHV无效， 对开发新的KSHV抗病毒药物的兴趣增加。一个有前途的新 抗病毒靶点是DNA聚合酶（Pol-8）和持续合成因子（PF-8） KCHV综合征PF与它们的同源DNA Pol相关联，使它们能够 合成延伸DNA而不与模板分离。的PF 已知某些其它疱疹病毒的基因是病毒DNA 合成和感染。事实上，只有少数dNTPs被整合到DNA中， Pol-8单独在体外，但当与PF-8组合时， incorporated.此外，Pol-8仅与PF-8复合和起作用，而不与PF-8复合和起作用。 其他疱疹病毒的PF。PF-8/Pol-8相互作用的特异性， 这是DNA合成所必需的，预测它应该是可能的， 鉴定能够阻断KSHV感染而不 扰乱正常的细胞活动我们的目标是阐明 PF-8/Pol-8进行性DNA合成，并开发和应用一种新的高 可用于鉴定抑制剂的通量筛选方法， 通过靶向PF-8/Pol-8特异性阻断DNA合成和KSHV感染。 目的是确定PF-8的亲和力、组成和结构 和Pol-8两者都在DNA上和在DNA上使用分析性超离心，表面 等离子体共振、诱变和晶体学。一种高通量测定， 将开发鉴定PF-8/Pol-8功能性抑制剂， 从NCI库中筛选化合物;使用基于 还将测试噬菌体展示和突变分析。所有 将测试体外阻断PF-8/Pol-8 DNA合成的抑制剂， 阻止KSHV感染的能力

DESCRIPTION: (Adapted from Applicant's Abstract) 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.