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

卡波西肉瘤相关疱疹病毒 (KSHV) 逃避自然杀伤细胞

• 批准号: 7613961
• 负责人: Alexis Spain Madrid
• 金额: $ 5.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7613961
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Antigen Presentation; Antiviral Agents; Apoptosis; B-Cell Lymphomas; Biological Assay; Blocking Antibodies; CD8B1 gene; Cancer Etiology; Cell Line; Cell surface; Cells; Coculture Techniques; Common Neoplasm; Cytolysis; Data; Disease; Down-Regulation; Flow Cytometry; HIV; Herpesviridae; Herpesviridae Infections; Human; Human Herpesvirus 8; Immune; Immune response; Immune system; Immunity; Immunocompromised Host; Immunofluorescence Immunologic; In Vitro; Individual; Infection; Interferon Type II; Interferons; Kaposi Sarcoma; Ligands; Lymphocyte; Lymphoma; MHC Class I Genes; Malignant Neoplasms; Measures; Mediating; Membrane Proteins; Messenger RNA; Methods; Monitor; Multicentric Angiofollicular Lymphoid Hyperplasia; NK Cell Activation; Natural Killer Cells; Organelles; Patients; Peripheral Blood Mononuclear Cell; Phenotype; Plasmids; Predisposition; Prevention; Proteins; Public Health; Screening procedure; Small Interfering RNA; Spain; Staining method; Stains; Surface; Testing; Transplant Recipients; Viral; Viral Proteins; Virus; cancer cell; cell growth; effusion; human disease; killer T cell; killings; knock-down; natural killer cell protein 44-kDa; novel; numb protein; prevent; protein phosphatase inhibitor-2; receptor; research study

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.

描述（由申请人提供）：Kaposi病毒与肉瘤相关的疱疹病毒（KSHV）可能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，它们在感染细胞上下调MHC-I，因此可能将这些细胞暴露于天然杀伤（NK）细胞介导的裂解中。我们假设KSHV还发展了一种逃避NK细胞杀死的机制。初步数据表明，在KSHV感染期间，一个NK激活配体NKP44-L被下调，并筛选了负责该活性的病毒基因产品，确定了蛋白质Kaposin B.该提案的长期目标是为了通过KSHV来表征KAPOSIN B的细胞，以表征这种新型的免疫逃避机制。在特定的目标1中，将检查Kaposin B介导的NKP44-L下调的机理。我们将执行NKP44-L的免疫荧光和细胞内流式细胞仪染色，以确定配体蛋白水平是否降低，以及在KSHV感染的和Kaposin b表达细胞中是否将蛋白质定位错误。对于特定目标2，为了确定Kaposin B是否是KSHV的活性，导致NKP44-L表面表达降低，使用siRNA在感染过程中将击倒Kaposin B mRNA。然后将对NKP44-L染色，以寻找下调的拯救。在特定目标3中，我们将通过进行体外NK激活和杀伤测定法测试Kaposin B下调NKP44-L下调的功能意义。表达Kaposin B或对照的靶细胞将与有和不具有NKP44表达的两个NKL细胞系共培养，以确定Kaposin B是否以NKP44依赖性方式使靶细胞脱敏nk杀死。这些实验还将使用原代人NK细胞进行，具有和不阻止针对NKP44的抗体。与公共卫生有关：KSHV是与免疫功能低下个体有关的几种人类疾病的病因，包括两个B细胞淋巴瘤和Kaposi的肉瘤（KS），这是未治疗的AIDS患者中最常见的恶性肿瘤。 NK细胞是淋巴细胞，在先天抗病毒免疫反应中特别重要。了解KSHV对NK细胞免疫的影响对于治疗和预防KSHV相关疾病很重要，并且可能会扩展到相关的疱疹病毒或其他癌症引起的病毒。