Functional analysis of the Epstein Barr virus nuclear antigen leader protein (EBNA-LP) in a viral context.

EB病毒（EBV）感染了大多数人。感染通常没有症状，但有些人在感染时会出现传染性单核细胞增多症（腺热）。这种病毒在某些白色血细胞中以休眠（潜伏）状态终生存在。然而，偶尔EBV会引起白色血细胞的癌症，特别是在个体也感染HIV或疟疾的情况下。EB病毒也与鼻咽癌有关，这在中国南方非常常见。总的来说，全世界约有1%的癌症被认为与EBV有关。建立EBV潜伏期的过程由一些病毒基因控制，包括EBV核抗原（EBNA）。这项研究资助的重点是EBNAs之一，EBNA-LP。以前的研究表明，EBNA-LP对于EBV将B细胞改变为更像癌症的状态（转化）的能力是重要的：在细胞培养中，EBV可以“转化”B细胞，使其无限分裂。EBNA-LP还被证明与许多细胞蛋白结合，并增强另一种EBNA EBNA 2激活某些基因的能力。然而，对于EBNA-LP在病毒感染背景下的作用还没有明确的认识。我们也不知道是否有任何报道的EBNA-LP与细胞蛋白的相互作用与其在EBV感染中的功能有关。为了克服这一点，我开发了新的方法来创建EBV毒株，其中EBNA-LP已被删除或工程改造为包含定义的突变。由于EBNA-LP基因的复杂性质，这是一个特殊的技术挑战。利用这些病毒，我们将能够评估EBNA-LP在B细胞转化中所起的作用，并鉴定由EBNA-LP控制的基因。我还将阐明EBNA-LP与细胞蛋白质相互作用的能力，确定EBNA-LP的哪些部分与这些蛋白质结合。通过关联EBNA-LP突变体与特定宿主蛋白相互作用的能力和转化中的特定缺陷，我将确定这些相互作用的生物学意义，并阐明哪些可以为EBV提供新的治疗靶点。参与相互作用的蛋白质界面和蛋白质修饰的位点可以为可以抑制这些相互作用的化合物提供靶标。准确揭示EBNA-LP对病毒的作用及其机制，将使未来开发新型抗EBV疗法的检测方法，从而消除病毒或EBV相关癌症的新型治疗方法。在癌症生物学和免疫逃避中有许多例子，其中通过研究已经进化为干扰细胞过程的病毒蛋白质发现了关键的细胞机制。因此，希望对疾病机制的更一般的见解也将来自本研究提案中的实验。

Epstein-Barr virus (EBV) infects most of the human population. Infection normally has no symptoms but some people develop infectious mononucleosis (glandular fever) when they become infected. The virus persists life-long in a dormant (latent) state in certain white blood cells. However, occasionally EBV causes cancers of the white blood cells, particularly where individuals are also infected by HIV or Malaria. EBV is also associated with a cancer of the nasopharynx which is unusually common in Southern China. In all, about 1% of all cancers worldwide are thought to involve EBV. The process of establishing EBV latency is governed by some of the viral genes, including the EBV nuclear antigens (EBNAs). The focus of this research grant is one of the EBNAs, EBNA-LP. Previous research has shown that EBNA-LP is important for the ability of EBV to change B cells into a more cancer-like state (transformation): in cell culture EBV can 'transform' B cells to divide indefinitely. EBNA-LP has also been shown to bind to a number of cellular proteins, and to enhance the abilities of another EBNA, EBNA2, to activate certain genes. However, there is no clear understanding of what EBNA-LP does in the context of virus infection. We also don't know whether any of the reported interactions of EBNA-LP with cell proteins are relevant to its functions in EBV infection.To overcome this, I have developed new methods to create EBV strains in which EBNA-LP has been deleted or engineered to contain defined mutations. This was an exceptional technical challenge due to the complex nature of the EBNA-LP gene. Using these viruses, we will be able to assess what roles are played by EBNA-LP in the transformation of B cells, and to identify genes that are controlled by EBNA-LP. I will also clarify the ability of EBNA-LP to interact with cellular proteins, identifying which parts of EBNA-LP bind to these proteins. By correlating the ability of EBNA-LP mutants to interact with a specific host protein and specific defects in transformation, I will identify the biological significance of these interactions and elucidate which ones may provide novel therapeutic targets for EBV. The protein interfaces involved in the interactions and the sites of protein modification may provide targets for chemical compounds that can inhibit those interactions. Revealing precisely what EBNA-LP does for the virus and its mechanism will thus allow future development of assays for novel anti-EBV therapies that may allow elimination of the virus or novel treatments of EBV associated cancers. There are many examples in cancer biology and immune evasion where key cell mechanisms have been discovered by investigation of virus proteins that have evolved to interfere with cell processes. It is therefore hoped that more general insights into disease mechanisms will also come out of the experiments in this research proposal.