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

描述(申请人提供)：卡波西氏肉瘤相关疱疹病毒(KSHV)病毒可导致人类持续感染和恶性细胞生长。KSHV编码两种蛋白，MIR1和MIR2，它们下调感染细胞上的MHC-I，从而可能使这些细胞暴露于自然杀伤细胞(NK)介导的裂解。我们推测，KSHV还进化出一种逃避NK细胞杀伤的机制。初步研究表明，KSHV感染过程中NK激活配体NKp44-L的表达下调，对该活性相关的病毒基因产物的筛选确定了Kaposin B蛋白。本研究的长期目标是研究Kaposin B的这种新的免疫逃避机制，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，我们将研究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感染细胞和Kaposin B表达细胞中的配体蛋白水平是否降低，以及该蛋白是否错误定位。对于特定的目的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，将在感染过程中利用siRNA敲除Kaposin BmRNA。然后对细胞进行NKp44-L染色，以寻找下调调控的补救措施。在具体目标3中，我们将通过体外NK激活和杀伤实验来检验Kaposin B下调NKp44-L的功能意义。将表达Kaposin B的靶细胞或对照细胞与两个表达NKp44和不表达NKp44的NKL细胞株共同培养，以确定Kaposin B是否以NKp44依赖的方式使靶细胞对NK杀伤不敏感。这些实验也将使用原代人类NK细胞，在有和没有阻断NKp44抗体的情况下进行。与公共卫生相关：KSHV是几种与免疫功能受损的人有关的人类疾病的病原体，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细胞免疫的影响对于治疗和预防KSHV相关疾病具有重要意义，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.