Enhanced Cell-mediated Immunogenicity of KSHV LANA1 Protein

描述（由申请人提供）：细胞免疫监测在控制卡波西肉瘤相关疱疹病毒（KSHV/HHV8）相关恶性肿瘤中的重要性反映在KSHV/HHV8感染的免疫抑制（艾滋病和移植）患者的高发病率中。在KS和原发性积液性淋巴瘤（PEL）肿瘤中，病毒在很大程度上是潜伏的，允许大多数病毒蛋白逃避免疫效应的监视。然而，潜伏期相关核抗原1 （LANA1）必须表达才能使病毒在潜伏期存活，因此是消除KSHV库感染的理想疫苗靶点。LANA1与eb病毒（EBV）的EBNA1蛋白一样，具有抑制LANA1特异性细胞毒性淋巴细胞（CTL）反应的分子特性。在蛋白质合成或周转过程中，病毒蛋白通过蛋白酶体降解被加工成CD8+细胞识别的表位。合成过程中的降解通过细胞对蛋白质错误折叠的识别（缺陷核糖体产物（DRiPs））发生。我们在这里表明，LANA1蛋白具有降低成熟蛋白周转率和新生蛋白翻译率的特定序列特征。与其他细胞和病毒蛋白相比，LANA1具有显著增强的蛋白稳定性，并且用蛋白酶体抑制剂预处理不会导致其积累。LANA1内部重复结构域的缺失导致LANA1合成速率的显著增加。因此，LANA1的主要结构特征可能通过减少成熟蛋白的周转和通过延迟多肽翻译确保最小的DRiP呈现来限制LANA1肽的MHC I呈递。我们的建议旨在定义这些过程，并确定它们对LANA1特异性CTL生成的重要性。具体目标I和II将通过精细定位，确定参与成熟蛋白周转和合成迟缓的LANA1结构域。为了检验降解抑制（Dl），我们将测量LANA1衍生物的泛素化和周转。假定的Dl结构域将被克隆到具有定义的降解动力学的异源蛋白中，以便测定降解过程的特定步骤。此外，我们将设计强蛋白酶体降解信号到LANA1，以确定是否可以克服Dl功能。同样，为了检查合成阻滞（SR），将测量LANA1及其衍生物的DRiP形成的定量。将评估SR的具体机制，如罕见密码子的使用。在Specific Aim III中，我们将使用LANA1和成熟的鸡卵白蛋白模型直接评估LANA1降解抑制和合成阻滞对小鼠CDS免疫应答的影响。这些结果将为有效的抗潜伏期KSHV疫苗奠定基础。

DESCRIPTION (provided by applicant): The importance of cellular immune surveillance in controlling Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8)-related malignancies is reflected by the high rate of disease among immunosuppressed (AIDS and transplant) patients who are KSHV/HHV8 infected. In KS and primary effusion lymphoma (PEL) tumors, the virus is largely latent allowing most viral proteins to escape immune effector surveillance. The latency-associated nuclear antigen 1 (LANA1), however, must be expressed for viral survival during latency and therefore represents an ideal vaccine target to eliminate KSHV reservoir infection. LANA1, like the EBNA1 protein of Epstein Barr virus (EBV), has molecular characteristics that inhibit generation LANA1-specific cytotoxic lymphocyte (CTL) responses. Viral proteins are processed into CD8+ cell-recognized epitopes through proteasomal degradation during protein synthesis or turnover. Degradation at synthesis occurs through cellular recognition of protein misfolding (defective ribosomal products (DRiPs)). We show here that LANA1 protein has specific sequence features that decrease rates of both mature protein turnover and nascent protein translation. LANA1 has markedly enhanced protein stability compared to other cellular and viral proteins and pretreatment with proteasome inhibitors does not lead to its accumulation. Deletion of LANA1 internal repeat domains causes a pronounced increase in LANA1 synthesis rate. Thus, primary LANA1 structural features may limit MHC I presentation of LANA1 peptides by diminishing mature protein turnover and ensuring minimal DRiP presentation through retarded polypeptide translation. Our proposal seeks to define these processes and to determine their significance to LANA1 specific CTL generation. Specific Aims I and II will define, through fine-mapping, the LANA1 domains involved in mature protein turnover and synthesis retardation. To examine degradation-inhibition (Dl), we will measure ubiquitinylation and turnover of LANA1 derivatives. The putative Dl domain will be cloned into heterologous proteins having defined degradation kinetics in order to assay specific steps of the degradation process. In addition, we will engineer strong proteasome-degradation signals into LANA1 to determine if the Dl function can be overcome. Similarly, to examine synthesis-retardation (SR), quantitation of DRiP formation for LANA1 and derivatives will be measured. Specific mechanisms for SR, such as rare codon usage, will be assessed. In Specific Aim III, we will directly assess the impact of LANA1 degradation-inhibition and synthesis-retardation on CDS immune responses in mice using LANA1 and a well-established chicken ovalbumin model. These results will lay the groundwork for an effective anti-latency KSHV vaccine.