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

描述（由申请人提供）：病毒卡波西肉瘤相关疱疹病毒（KSHV）可引起人类持续感染和恶性细胞生长。KSHV编码两种蛋白，MIR1和MIR2，下调感染细胞上的mhc - 1。因此，这些细胞可能暴露于自然杀伤（NK）细胞介导的裂解。我们假设KSHV也进化出一种机制来逃避NK细胞的杀伤。初步数据显示，一种NK激活配体NKp44-L在KSHV感染期间下调，对负责该活性的病毒基因产物的筛选鉴定出了Kaposin B蛋白。本研究的长期目标是表征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Specific Aim 1中，我们将探讨Kaposin b介导的NKp44-L下调的机制。我们将对NKp44-L进行免疫荧光和细胞内流式细胞术染色，以确定kshv感染和表达Kaposin b的细胞中配体蛋白水平是否降低，以及蛋白是否错定位。对于Specific Aim 2，为了确定Kaposin B是否是KSHV中导致NKp44-L表面表达下降的活性，将在感染期间使用siRNA敲低Kaposin B mRNA。然后对细胞进行NKp44-L染色，以寻找对下调的拯救。在Specific Aim 3中，我们将通过体外NK激活和杀伤实验来测试Kaposin B下调NKp44-L的功能意义。将表达Kaposin B或对照的靶细胞与两株表达和不表达NKp44的NKL细胞系共培养，以确定Kaposin B是否以依赖NKp44的方式使靶细胞对NK杀伤脱敏。这些实验也将使用原代人NK细胞进行，具有或不具有针对NKp44的阻断抗体。与公共卫生的相关性：KSHV是与免疫功能低下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氏肉瘤（KS），这是未经治疗的艾滋病患者中最常见的恶性肿瘤。NK细胞是在先天抗病毒免疫反应中特别重要的淋巴细胞。了解KSHV对NK细胞免疫的影响对KSHV相关疾病的治疗和预防具有重要意义，并可能延伸到相关的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.