T-cell Transformation by Oncoviruses

在本项目中，我们有几个目标：目标1：将异位表达的HTLV-1非结构蛋白的功能生化研究所获得的信息与HTLV-1在体外相关细胞模型(树突状细胞和T细胞)中的感染性和持久性相结合。目的：将异位表达的HTLV-1非结构蛋白的功能生化研究结果与HTLV-1在相关动物模型中的感染性和持久性结合起来。病毒基因组编码几种非结构蛋白的mRNAs，这些蛋白影响细胞途径并调节病毒复制。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的蛋白水解性决定了其在细胞内的定位和功能。去除p12氨基末端的非规范内质网(ER)滞留/恢复信号是运输到高尔基体并产生完全切割的8 kDa蛋白所必需的。这个8 kDa的蛋白运输到细胞表面，在T细胞受体(TCR)连接后被招募到免疫突触，并下调TCR近端信号。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粘连蛋白相互作用。对HTLV-1感染患者体外样本中ORF-I的基因分析显示，ORF-I中频繁的氨基酸替换抑制了蛋白水解性切割，提示p12I的ER相关功能可能是在体内选择的。我们计划使用反向遗传方法和动物模型来了解orf-I变异对维持宿主病毒载量的贡献。HTLV-1orf II编码p30II，这是一种核/核仁蛋白，不仅通过转录后机制调节病毒的表达，而且还影响与宿主反应有关的基因的表达，如TLR-4。此外，orf II编码p13，这是一种我们最近证明通过靶向和降解病毒转录激活因子Tax来减少病毒复制的小蛋白。我们最近证明了p13是病毒表达的负调控因子。单独表达，p13定位于线粒体，但在Tax存在的情况下，部分p13被泛素化、稳定并重新路由到核斑点。P13蛋白直接与Tax结合，减少Tax与CBP/p300转录共激活子的结合，并通过降低Tax转录活性抑制病毒表达。我们已经产生了不表达p30和/或p13的感染性HTLV-1分子克隆。我们正在对这些病毒进行体外和体内测试。我们发现，P30对HTLV-1在单核细胞来源的树突状细胞中的复制能力有很大的影响，是猕猴持续感染所必需的。进一步的研究正在进行中，以确定P30和P13对体内传染性的影响正在动物模型中进行测试。

In this project we have several aims: Aim 1: To integrate information obtained by functional biochemical studies on ectopically expressed non-structural HTLV-1 proteins to infectivity and persistence of HTLV-1 in relevant cellular models in vitro (dendritic cells and T-cells). Aim 2: To integrate information obtained by functional biochemical studies on ectopically expressed non-structural HTLV-1 proteins to infectivity and persistence of HTLV-1 in relevant animal models of HTLV-1 infection. 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 and down-regulates TCR proximal signaling. 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. We plan to use reverse genetic methods and animal models to understand the contribution of orf-I variants to the maintenance of viral load in the host. The HTLV-1 orf II encodes p30II, a nuclear/nucleolar protein that not only regulates viral expression by a post-transcriptional mechanism, but also affects the expression of genes involved in host responses such as TLR-4. In addition orf II encodes p13, a small protein that we have recently demonstrate to decrease viral replication by targeting and degrading Tax, the viral transcriptional activator. We have recently demonstrated that p13 is a negative regulator of virus expression. Expressed separately, p13 localizes to the mitochondria, but in the presence of Tax, part of it is ubiquitinated, stabilized, and re-routed to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional co-activator and, by reducing Tax transcriptional activity, suppresses viral expression. We have generated infectious HTLV-1 molecular clones that do not express p30 and/or p13. We are testing these viruses both in vitro and in vivo. We have found that p30 affects dramatically the ability of HTLV-1 to replicate in monocytoid-derived dendritic cells and is necessary for persistence of infection in macaques. Further studies are ongoing to determine the effect of p30 and p13 on in vivo infectivity is being tested in animal models.