Kaposi's Sarcoma-Associated Herpesvirus (KSHV) evasion of Natural Killer cells

描述（由申请方提供）：卡波西肉瘤相关疱疹病毒（KSHV）可导致人类持续感染和恶性细胞生长。KSHV编码两种蛋白质MIR 1和MIR 2，其下调感染细胞上的MHC-I，从而潜在地将这些细胞暴露于自然杀伤（NK）细胞介导的裂解。我们假设KSHV也进化出一种逃避NK细胞杀伤的机制。初步数据显示，一种NK活化配体NKp 44-L在KSHV感染期间下调，并且对负责该活性的病毒基因产物的筛选鉴定出蛋白Kaposin B。该提案的长期目标是表征KSHV的这种新型免疫逃避机制，并确定Kaposin B是否允许KSHV感染的细胞逃避NK细胞的裂解。在具体目标1中，将检查Kaposin B介导的NKp 44-L下调的机制。我们将对NKp 44-L进行免疫荧光和细胞内流式细胞术染色，以确定在KSHV感染和Kaposin B表达细胞中配体蛋白水平是否降低，以及蛋白质是否错误定位。对于特异性目的2，为了确定Kaposin B是否是KSHV中导致NKp 44-L表面表达降低的活性，在感染期间使用siRNA敲低Kaposin B mRNA。然后对细胞进行NKp 44-L染色以寻找下调的拯救。在具体目标3中，我们将通过进行体外NK活化和杀伤测定来测试Kaposin B下调NKp 44-L的功能意义。将表达Kaposin B或对照的靶细胞与表达和不表达NKP 44的两种NKL细胞系共培养，以确定Kaposin B是否以NKP 44依赖性方式使靶细胞对NK杀伤脱敏。这些实验也将使用原代人NK细胞进行，有和没有针对NKp 44的阻断抗体。与公共卫生的相关性：KSHV是与免疫受损个体相关的几种人类疾病的病原体，包括两种B细胞淋巴瘤和卡波西肉瘤（KS），卡波西肉瘤是未经治疗的AIDS患者中最常见的恶性肿瘤。NK细胞是在先天性抗病毒免疫应答中特别重要的淋巴细胞。了解KSHV对NK细胞免疫的影响对于治疗和预防KSHV相关疾病非常重要，并且可能潜在地扩展到相关疱疹病毒或其他致癌病毒。

DESCRIPTION (provided by applicant): The virus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) can cause persistent infection and malignant cell growth in humans. KSHV encodes two proteins, MIR1 and MIR2, that downregulate MHC-I on infected cells, .thus potentially exposing these cells to Natural Killer (NK) cell mediated lysis. We hypothesized that KSHV has also evolved a mechanism to evade killing by NK cells. Preliminary data show that one NK activating ligand, NKp44-L, is downregulated during KSHV infection, and screening for a viral gene product responsible for this activity identified the protein Kaposin B. The long-term objective of this proposal is to characterize this novel immune evasion mechanism by KSHV, and to determine whether Kaposin B allows KSHV-infected cells to evade lysis by NK cells. In Specific Aim 1, the mechanism of Kaposin B-mediated downregulation of NKp44-L will be examined. We will perform immunofluorescence and intracellular flow cytometric staining of NKp44-L, in order to determine whether ligand protein levels are decreased, and whether the protein is mislocalized, in KSHV-infected and Kaposin B-expressing cells. For Specific Aim 2, in order to determine whether Kaposin B is the activity in KSHV that causes decreased NKp44-L surface expression, Kaposin B mRNA will be knocked down during infection using siRNA. Cells will then be stained for NKp44-L to look for rescue of the downregulation. In Specific Aim 3, we will test the functional significance of NKp44-L downregulation by Kaposin B by performing in vitro NK activation and killing assays. Target cells expressing Kaposin B or a control will be co-cultured with two NKL cell lines, with and without NKp44 expression, to determine whether Kaposin B desensitizes target cells to NK killing in an NKp44-dependent manner. These experiments will also be performed using primary human NK cells, with and without blocking antibodies against NKp44. Relevance to public health: KSHV is a causative agent of several human diseases associated with immunocompromised individuals, including two B-cell lymphomas and Kaposi's Sarcoma (KS), the most common malignancy in untreated AIDS patients. NK cells are lymphocytes that are particularly important in the innate anti-viral immune response. Understanding the effects on NK cell immunity by KSHV is important for the treatment and prevention of KSHV-related diseases, and may potentially extend to related herpesviruses or other cancer- causing viruses.