VIRAL SMALL RNPS AND CELL TRANSFORMATION

（申请人描述）正在研究在两种疱疹病毒转化的淋巴样细胞中发现的小核RNA-蛋白（snRNP）复合物的作用。爱泼斯坦-巴尔病毒感染并转化人类B细胞，是传染性单核细胞增多症的病原体，也与几种人类癌症有关。猴疱疹病毒在新世界猴体内诱发致命的淋巴瘤和白血病，在新世界猴体内转化人类和猴子的T淋巴细胞和白血病，在培养物中转化人类和猴子的T淋巴细胞。在转化细胞中表达的少数病毒基因产物是2个ebv编码的eber和7个猴血蜱编码的HSURs。这些小的核rna是丰富的，保守的，并与宿主自身抗原相关联，宿主自身抗原是细胞snRNPs的组成部分。然而，eber和hsus对于病毒的转化生长都不是必需的。提出的实验基于这样的假设，即eber和hsur通过隔离重要的宿主蛋白来扰乱细胞代谢，从而促进转化。EBER1结合宿主核糖体蛋白L22的显著部分，其参与一些急性髓性白血病患者的染色体易位。EBER1与L22相互作用的后果将通过定位L22在60S核糖体中的结合位点来研究，并询问L22缺失的核糖体是否会增强核抗原（EBNA） mrna上游AUGs的读通透性，从而增加其在潜伏期的翻译。hsur1、2和5在其5'端有AU3A基序结合HuR， HuR是一种宿主蛋白，其过表达稳定了原癌基因、细胞因子和淋巴因子的正常短寿命mrna。HUR的核-细胞质穿梭基序及其与几种蛋白磷酸酶2A （PP2A）抑制剂的相互作用，其中一种被报道具有肿瘤抑制活性，将通过突变分析进行解剖。au3a定向RNA衰变的亚细胞位置以及HuR和hnRNPD在MRNA稳定性中发挥相反作用的可能性将被研究。将研究人乳头瘤病毒E6/E7癌蛋白的表达，以确定PP2A抑制剂和HuR是否是调节mRNA稳定性的磷酸化级联的组成部分。我们将在爪蟾系统中研究au3a导向的mRNA死基化/衰变机制及其在发育早期的调控。EBER2和hsur3、4、6和7 snRNPs的功能将通过鉴定相互作用蛋白来寻找线索。

DESCRIPTION: (Applicant's Description) The roles of small nuclear RNA- protein (snRNP) complexes found in lymphoid cells transformed by two herpesviruses are being investigated. Epstein-Barr virus infects and transforms human B cells and is the causative agent of infectious mononucleosis, as well as being associated with several human cancers. Herpesvirus saimiri induces fatal lymphomas and leukemias in New World monkeys and transforms human and monkey T lymphocytes and leukemias in New World monkeys and transforms human and monkey T lymphocytes in culture. Among the few viral gene products expressed in transformed cells are the two EBV-encoded EBERs and the seven H. saimiri-encoded HSURs. These small nuclear RNAs are abundant, conserved, and associate with host auto-antigens that are components of cellular snRNPs. Yet, both EBERs and HSURs are non-essential for viral growth of transformation. Proposed experiments are based on the hypothesis that EBERs and HSURs perturb cellular metabolism by sequestering important host proteins, thereby facilitating transformation. EBER1 binds a significant fraction of the host's ribosomal protein L22, whose is involved in chromosomal translocations in some patients with acute myeloid leukemia. Consequences of EBER1 interaction with L22 will be studied by locating the L22 binding site in the 60S ribosome and asking whether L22-depleted ribosomes enhance the read-through of upstream AUGs in nuclear antigen (EBNA) mRNAs to increase their translation during latency. HSURs 1, 2, and 5 have AU3A motifs at their 5' ends that bind HuR, a host protein whose over-expression stabilizes the normally short-lived mRNAs for proto-oncogenes, cytokines and lymphokines. HUR's nucleo- cytoplasmic shuttling motif and its interactions with several protein phosphatase 2A (PP2A) inhibitors, one of which is reported to have tumor suppressor activity will be dissected by mutational analyses. The subcellular location of AU3A-directed RNA decay and the possibility that HuR and hnRNPD play opposing roles in MRNA stability will be investigated. The expression of human papillomavirus E6/E7 oncoproteins will be studied to establish whether the PP2A inhibitors and HuR are components of a phosphorylation cascade regulating mRNA stability. The mechanism of AU3A-directed mRNA deadenylation/decay and its regulation during early development will be studied in the Xenopus system. Clues to the functions of the EBER2 and HSUR 3, 4, 6 and 7 snRNPs will be sought by identifying interaction proteins.