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

爱泼斯坦-巴尔病毒(EBV)感染人类的大部分人群。感染通常没有症状，但有些人感染后会发展成传染性单核细胞增多症(腺热)。该病毒在某些白血球中以潜伏状态持续终生。然而，EBV偶尔会导致白细胞癌症，特别是在个人也感染艾滋病毒或疟疾的情况下。EB病毒还与鼻咽癌有关，鼻咽癌在南方中国非常常见。总体而言，全世界约1%的癌症被认为与EBV有关。建立EBV潜伏期的过程受一些病毒基因的控制，包括EBV核抗原(EBNAs)。这项研究拨款的重点是EBNAs之一，EBNA-LP。以前的研究表明，EBNA-LP对于EBV将B细胞转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，EBNA2，激活某些基因的能力。然而，对于EBNA-LP在病毒感染的背景下所做的工作还没有明确的了解。我们也不知道是否有任何已报道的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已被删除或被改造为包含明确的突变。由于EBNA-LP基因的复杂性，这是一项特殊的技术挑战。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，并识别由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。通过将EBNA-LP突变体与特定宿主蛋白相互作用的能力与转化中的特定缺陷相关联，我将确定这些相互作用的生物学意义，并阐明哪些可能为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置可能为抑制这些相互作用的化合物提供靶标。准确地揭示EBNA-LP对病毒的作用及其机制将使未来能够开发新的抗EBV疗法的分析，从而可能消除病毒或EBV相关癌症的新疗法。在癌症生物学和免疫逃避中，有许多例子是通过对病毒蛋白的研究发现了关键的细胞机制，这些病毒蛋白已经进化到干扰细胞过程。因此，希望这项研究提案中的实验也能对疾病机制有更全面的了解。

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.