T-cell Transformation by Oncoviruses

在这个项目中，我们有两个主要的方法：方法1：将通过功能生化研究获得的信息整合到HTLV-1在体外树突状细胞和t细胞中的感染性和持久性。方法2：在动物模型中验证orf I和orf II在病毒持久性中的作用。病毒基因组编码几种影响细胞通路和调节病毒复制的非结构蛋白的mrna。其中一种蛋白质p12由orf I编码，定位于内质网、高尔基体和细胞膜。p12的蛋白水解裂解决定了其细胞定位和功能。去除p12氨基末端的非规范内质网（ER）保留/恢复信号是运输到高尔基体和产生完全裂解的8kda蛋白所必需的。8 kDa蛋白运输到细胞表面，在t细胞受体（TCR）结扎后被募集到免疫突触，下调TCR近端信号并增加病毒传播。p12的全长形式存在于内质网中，并与白细胞介素-2受体（IL-2R）的β链和γ -c链、主要组织相容性复合体（MHC） I类的重链以及钙网蛋白和钙连蛋白相互作用。htlv -1感染患者离体样本的ORF-I遗传分析显示ORF-I中频繁的氨基酸替换抑制蛋白水解裂解，提示p12I的ER相关功能可能在体内被选择。由于HTLV-1或f- 1对病毒的传播和持续很重要，我们试图确定HTLV-1感染个体中p12蛋白的遗传变异，并研究p12突变、其切割状态、前病毒载量和疾病结局之间的可能关联。在这项研究中，我们对160名患者进行了反向遗传学研究，以确定orf-I的突变。我们发现，与主要表达p12或主要表达p8的克隆相比，携带同时表达p12和p8的克隆的个体具有更高的病毒载量。此外，我们构建了表达不同orf-I亚型的感染性分子克隆（p12/p8;p12;p8）。克隆细胞系建立、鉴定并应用于恒河猴模型。这些研究的结果清楚地表明，病毒的持久性需要p12和p8亚型的表达。重要的是，我们的结果表明p12/p8表达影响CTL逃逸。我们还分析了在p12和p8功能中的作用。我们发现HTLV-1 p12和p8形成二硫连接的同型和异型二聚体，这些蛋白的单体形式是棕榈酰化的。这些蛋白中半胱氨酸39的突变破坏了p12和p8的二聚化和棕榈酰化，但不影响蛋白的定位。这些研究表明p8的棕榈酰化破坏了二聚体的形成，并以单体形式为目标，调节细胞信号通路，增加细胞间的粘附和病毒感染。确定p12和p8二聚化和功能调控的机制将促进我们对HTLV-1发病机制的理解，并可能为治疗HTLV-1感染者找到潜在的新治疗靶点。

In this project we have two main approaches: Approach 1: To integrate information obtained by functional biochemical studies to infectivity and persistence of HTLV-1 in dendritic cells and T-cells in vitro. Approach 2: To verify the role of orf I and orf II in viral persistence in animal models. The viral genome encodes mRNAs for several non-structural proteins that affect cellular pathways and modulate viral replication. One such protein, p12, encoded by orf I, localizes to the ER and Golgi and cellular membranes. The proteolytic cleavage of p12 dictates its cellular localization and functions. The removal of a non-canonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12 is necessary for trafficking to the Golgi apparatus and the generation of a completely cleaved 8 kDa protein. The 8 kDa protein traffics to the cell surface, is recruited to the immunological synapse following T-cell receptor (TCR) ligation, down-regulates TCR proximal signaling and increases viral transmission. The full length form of p12 resides in the ER and interacts with the beta and gamma-c chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals frequent amino acid substitutions within orf-I that inhibit proteolytic cleavage, suggesting that ER associated functions of p12I may be selected in vivo. Because HTLV-I orf-I is important for viral transmission and persistence, we sought to determine the genetic variation of p12 protein in HTLV-1 infected individuals and investigate a possible association between p12 mutations, its cleavage status, proviral load, and disease outcome. In this study, we performed reverse genetics of 160 patients to identify mutations in orf-I. We found that individuals that harbor clones expressing both p12 and p8 have higher virus loads compared to individuals that harbor clones expressing predominantly p12 or predominantly p8. Further, we constructed infectious molecular clones expressing distinct orf-I isoforms (p12/p8;p12;p8). Clonal cell lines were established, characterized and used in a rhesus macaque model. Results from these studies clearly demonstrate that viral persistence requires expression of both p12 and p8 isoforms. Importantly, our results suggest that p12/p8 expression impacts CTL escape.We also analyzed the role of in p12 and p8 function. We found that HTLV-1 p12 and p8 form disulfide-linked homo-and heterodimers and that the monomeric forms of these proteins are palmitoylated. Mutation of cysteine 39 within these proteins disrupted dimerization and palmitoylation of both p12 and p8 without affecting protein localization. These studies suggest palmitoylation of p8 dirupts dimer formation and targets the monomeric form to modulate cell signaling pathways to increase cell-to-cell adhesion and viral infectivity. Determining the mechanism by which p12 and p8 dimerization and functions are regulated would advance our understanding of HTLV-1 pathogenesis and could identify potential novel therapeutic targets for the treatment of HTLV-1-infected individuals.