T-cell Transformation by Oncoviruses

在这个项目中，我们有三种主要的方法。方法1:p12衍生的裂解产物p8运输到细胞表面，减少TCR信号，增加病毒传播。在转染的细胞中发现p8和p12的平衡产生。我们假设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感染的个体中，我们发现orf- 1的多态性与切割减少有关，29位甘氨酸到丝氨酸（G29S）导致p12多于p8，切割增加，26位天冬氨酸到天冬氨酸（D26N）导致p8多于p12，平衡生产，26位天冬氨酸（D26）导致p8和p12相似的表达。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26号位置有天冬氨酸。我们发现，与p8和p12的平衡产生相兼容的多态性患者的血液中病毒负担明显高于p8或p12较多的患者，这表明这两种蛋白质的产生对于建立和/或维持高病毒负担是最佳的。在HTLV-1分子克隆的orf-I中引入的突变G29S和D26N证实了p12和p8对于在猕猴中建立和维持HTLV-1感染是必要的。方法2：在体外和猕猴体内，切除orf-I和orf-II的表达会损害树突状细胞和单核细胞HTLV-1的复制。在触发细胞表面的TLR3/4后，p30抑制干扰素应答基因的转录，而不抑制内体的TLR7/8信号，而p8/p12同时影响TLR3/4和TLR7/8信号。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入和退出的必要条件，这些蛋白可能影响单核细胞/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，这反过来可能有利于维持高病毒负荷。对HTLV-1感染个体血液中分选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。我们发现中间CD14+CD16+，以及非经典CD14-CD16+单核细胞和病毒负荷呈正相关，特别是在TSP/HAM中。这些数据提示HTLV-1感染单核细胞可促进其向促炎单核细胞分化。我们将以THP-1为模型细胞系，在体外研究p8/p12和p30在趋化性、吞噬作用和细胞因子产生方面的影响。我们计划通过检测HTLV-1突变体感染的体外单核细胞和从不同水平病毒负荷的HTLV-1感染个体获得的体外感染单核细胞来扩展这些功能研究。方法3：我们假设猕猴的STLV-1感染可以作为HTLV-1感染的模型。我们发现，在STLV-1感染的猕猴中，中间和非经典单核细胞群体显著增加。未来的工作将评估这些单核细胞亚群是否也被STLV-1感染，以及该动物模型是否适合用于减少药物或免疫调节剂的病毒负担的测试策略。

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.