T-cell Transformation by Oncoviruses

肿瘤病毒对 T 细胞的转化

• 批准号: 8762999
• 负责人: Genoveffa Franchini
• 金额: $ 143.98万
• 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/8762999
• 关键词: 3' Splice Site; Ablation; Adult; Affect; Animal Model; Animals; Asparagine; Aspartic Acid; Binding; Blood; CD14 gene; CD3 Antigens; Calcium; Cell Line; Cell Nucleus; Cell physiology; Cell surface; Cells; Cleaved cell; Complex; Cytoplasm; DNA Polymerase II; Data; Dendritic Cells; Disease; EP300 gene; Endosomes; Equilibrium; Exons; FCGR3B gene; Future; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Glycine; Goals; Golgi Apparatus; HTATIP gene; Human T-lymphotropic virus 1; ICAM1 gene; ICAM3 gene; Immune; In Vitro; Individual; Infection; Inflammatory; Interferons; Interleukin 2 Receptor; Life; Ligation; Lysine; Lysosomes; Macaca; Maintenance; Mediating; Messenger RNA; Modeling; Molecular Cloning; Mutation; Nuclear; Nuclear Pore; Nuclear Protein; Oncornaviruses; Open Reading Frames; Oryctolagus cuniculus; Patients; Phagocytosis; Pharmaceutical Preparations; Plasmids; Population; Positioning Attribute; Production; Proline; Proteins; RNA; RNA Binding; RNA Splicing; Relative (related person); Retroviridae; Role; STAT5A gene; Serine; Signal Transduction; Simian T-lymphotropic virus 1; Sorting - Cell Movement; Surface; T-Cell Activation; T-Cell Leukemia; T-Cell Transformation; T-Lymphocyte; TLR3 gene; TLR7 gene; Taxes; Testing; Viral; Viral Genes; Viral Load result; Viral Proteins; Virus; Work; cofactor; cytokine; human disease; in vivo; leukemia/lymphoma; mRNA Export; mRNA Expression; monocyte; multicatalytic endopeptidase complex; mutant; prevent; research study; response; trafficking; transmission process; vacuolar H+-ATPase; viral DNA

In this project, we have three main approaches. Approach 1: The p12 derived cleavage product p8 traffics to the cell surface, decreases TCR signaling and increases virus transmission. A balanced production of p8 and p12 is found in transfected cells. We hypothesized that perturbation of the relative amount of p8 and p12 may affect HTLV-1 infectivity or persistence. In HTLV-1 infected individuals, we found polymorphisms in orf-I associated with a decreased cleavage, Glycine at position 29 to Serine (G29S) that results in more p12 than p8, increased cleavage, Aspartic acid at position 26 to Asparagine (D26N) that results in more p8 than p12 and balanced production, Aspartic acid at position 26 (D26), that results in similar expression of both p8 and p12. Most HTLV-1 infected people (33%) and the biologically active HTLV-1 molecular clones available have Aspartic acid at position 26. We found a significantly higher viral burden in the blood of patients with polymorphisms compatible with a balanced production of p8 and p12 than in those with more p8 or more p12, suggesting that the production of both proteins is optimal for the establishment and/or maintenance of high viral burden. Mutations G29S and D26N introduced within the orf-I of an HTLV-1 molecular clone confirmed that both p12 and p8 are necessary to establish and maintain HTLV-1 infection in macaques. Approach 2: Ablation of orf-I and orf-II expression impairs HTLV-1 replication in dendritic cells and monocytes in vitro and in macaques in vivo. p30 inhibits the transcription of Interferon-responsive genes following triggering of TLR3/4 on the cell surface but not of TLR7/8 signaling in the endosome, whereas, p8/p12 affects both TLR3/4 and TLR7/8 signaling. These data suggest that the inhibition of innate responses by these viral proteins is likely essential for virus entry and egress and that these proteins may affect monocyte/dendritic cell function. We hypothesized that HTLV-1 infection of monocytes in vivo alters monocyte function that, in turn, may favor maintenance of high virus burden. The analysis of sorted monocytes from the blood of HTLV-1 infected individuals demonstrated various levels of viral DNA within the monocyte subsets. We found that there was a positive correlation with intermediate CD14+CD16+, as well as, non-classical CD14-CD16+ monocytes and virus burden, particularly in TSP/HAM. These data suggest that HTLV-1 infection of monocytes may promote their differentiation to pro-inflammatory monocytes. We will investigate the effects of p8/p12 and p30 using the whole virus or as individual proteins on chemo-taxis, phagocytosis and cytokine production using THP-1 as a model cell line in vitro. We plan to extend these functional studies by examining in vitro monocytes infected with HTLV-1 mutants and ex vivo infected monocytes obtained from HTLV-1 infected individuals with various levels of viral burden. Approach 3: We hypothesized that STLV-1 infection of macaques could be exploited as a model of HTLV-1 infection. We found that in STLV-1 infected macaques there is a significant increase in intermediate and non-classical monocyte populations. Future work will assess whether these monocyte subsets are also infected by STLV-1 and whether this animal model is suitable for testing strategies to decrease viral burden with drugs or immune modulators.

在这个项目中，我们有三个主要方法。途径1：p12裂解产物p8进入细胞表面，减少TCR信号转导，增加病毒传播。在转基因细胞中发现了p8和p12的平衡产生。我们推测，p8和p12相对量的扰动可能会影响HTLV-1的传染性或持久性。在HTLV-1感染者中，我们发现orf-I的多态与切割减少有关，29位甘氨酸到丝氨酸(G29S)导致p12多于p8，切割增加，26位天冬氨酸导致p8多于p12(D26N)，导致p8多于p12和平衡产生，天冬氨酸在26位(D26)，导致p8和p12的相似表达。大多数HTLV-1感染者(33%)和现有的具有生物活性的HTLV-1分子克隆在第26位含有天冬氨酸。我们发现，与p8和p12的平衡产生相适应的多态患者的血液中的病毒负荷显著高于那些p8或p12以上的患者，这表明这两种蛋白的产生对于建立和/或维持高病毒负荷是最佳的。在HTLV-1分子克隆的orf-I中引入的G29S和D26N突变证实了p12和p8都是建立和维持猕猴感染HTLV-1所必需的。方法2：去除orf-I和orf-II的表达会在体外和体内损害树突状细胞和单核细胞中HTLV-1的复制。P30抑制细胞表面TLR3/4信号转导后干扰素反应基因的转录，但不抑制内体TLR7/8信号转导，而p8/p12则同时影响TLR3/4和TLR7/8信号转导。这些数据表明，这些病毒蛋白对先天反应的抑制可能是病毒进出所必需的，这些蛋白可能会影响单核细胞/树突状细胞的功能。我们假设HTLV-1在体内感染单核细胞会改变单核细胞的功能，进而可能有利于维持高病毒负荷。对HTLV-1感染者血液中分离的单核细胞的分析表明，单核细胞亚群中存在不同水平的病毒DNA。研究发现，TSP/HAM病毒载量与CD14+CD16+、非经典CD14-CD16+单核细胞呈正相关。提示HTLV-1感染单核细胞可能促进其向促炎单核细胞分化。我们将以THP-1细胞为模型细胞，研究p8/p12和p30蛋白在体外对THP-1细胞趋化、吞噬和细胞因子产生的影响。我们计划通过检测感染HTLV-1突变体的体外单核细胞和从不同病毒载量的HTLV-1感染者获得的体外感染单核细胞来扩展这些功能研究。方法3：我们假设STLV-1感染猕猴可以作为HTLV-1感染的模型。我们发现，在感染STLV-1的猕猴中，中间单核细胞和非经典单核细胞数量显着增加。未来的工作将评估这些单核细胞亚群是否也感染了STLV-1，以及这种动物模型是否适合测试药物或免疫调节剂降低病毒负担的策略。