T-cell Transformation by Oncoviruses

肿瘤病毒对 T 细胞的转化

• 批准号: 8348887
• 负责人: Genoveffa Franchini
• 金额: $ 178.67万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348887
• 关键词: Adult; Affect; Amino Acid Substitution; Animal Model; Binding; Biochemical; Calnexin; Cell model; Cell surface; Cellular Membrane; Chronic; Cleaved cell; Complex; Dendritic Cells; Disease; EP300 gene; Endoplasmic Reticulum; Excision; Gene Expression; Generations; Goals; Golgi Apparatus; Human; Human T-lymphotropic virus 1; Immune response; In Vitro; Individual; Infection; Interleukin 2 Receptor; Length; Life; Ligation; Macaca; Maintenance; Major Histocompatibility Complex; Messenger RNA; Methods; Mitochondria; Molecular Cloning; Nuclear; Nucleolar Proteins; Oncornaviruses; Open Reading Frames; Pathway interactions; Patients; Proteins; Recruitment Activity; Retrieval; Retroviridae; Sampling; Signal Transduction; T-Cell Leukemia; T-Cell Receptor; T-Cell Transformation; T-Lymphocyte; Taxes; Testing; Transcription Coactivator; Variant; Viral; Viral Genes; Viral Genome; Viral Load result; Viral load measurement; Virus; Virus Diseases; calreticulin; genetic analysis; human disease; immunological synapse; in vivo; leukemia; leukemia/lymphoma; positional cloning; prevent; protein transport; therapeutic target; trafficking

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.

在这个项目中，我们有几个目标：目标1：整合信息的功能生化研究异位表达的非结构HTLV-1蛋白的感染性和持久性的HTLV-1在体外相关的细胞模型（树突状细胞和T细胞）。目标二：整合通过异位表达的非结构性HTLV-1蛋白的功能生化研究获得的信息，以确定HTLV-1在相关HTLV-1感染动物模型中的感染性和持久性。病毒基因组编码影响细胞途径和调节病毒复制的几种非结构蛋白的mRNA。一种这样的蛋白质，p12，由orf I编码，定位于ER和高尔基体和细胞膜。p12的蛋白水解切割决定了其细胞定位和功能。在p12的氨基末端内去除非典型内质网（ER）保留/检索信号对于运输到高尔基体和产生完全切割的8 kDa蛋白是必要的。该8 kDa蛋白运输至细胞表面，在T细胞受体（TCR）连接后被募集至免疫突触，并下调TCR近端信号传导。p12的全长形式存在于ER中，并与白细胞介素-2受体（IL-2 R）的β和γ-c链、I类主要组织相容性复合体（MHC）的重链以及钙网蛋白和钙连接蛋白相互作用。HTLV-1感染患者的离体样本的ORF-I的遗传分析揭示了ORF-I内抑制蛋白水解裂解的频繁氨基酸取代，表明ER相关的p12 I功能可以在体内选择。我们计划使用反向遗传学方法和动物模型来了解orf-I变异体对维持宿主病毒载量的贡献。HTLV-1 orf II编码p30 II，p30 II是一种核/核仁蛋白，不仅通过转录后机制调节病毒表达，而且还影响参与宿主反应的基因（如TLR-4）的表达。此外，orf II编码p13，这是一种小蛋白，我们最近证明它通过靶向和降解病毒转录激活因子Tax来减少病毒复制。我们最近证明，p13是一个负调节病毒表达。单独表达时，p13定位于线粒体，但在Tax的存在下，它的一部分被泛素化，稳定化，并重新路由到核斑点。p13蛋白直接结合Tax，降低Tax与CBP/p300转录辅激活因子的结合，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。我们正在体外和体内测试这些病毒。我们发现p30显著影响HTLV-1在单核细胞衍生的树突状细胞中复制的能力，并且是猕猴中持续感染所必需的。进一步的研究正在进行中，以确定p30和p13对体内感染性的影响，并在动物模型中进行测试。