Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation.
Conserved Motifs within Hepatitis C Virus Envelope (E2) RNA and Protein Independently Inhibit T Cell Activation.
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DOI：
10.1371/journal.ppat.1005183
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发表时间：
2015-09
期刊：
影响因子：
6.7
通讯作者：
Stapleton JT
中科院分区：
文献类型：
--
作者：
Bhattarai N;McLinden JH;Xiang J;Kaufman TM;Stapleton JT
T细胞受体（TCR）信号传导是T细胞活化、增殖、分化和效应子功能所需的。丙型肝炎病毒（HCV）感染与T细胞功能受损相关，导致持续的病毒血症、延迟和不一致的抗体应答以及轻度免疫功能障碍。虽然多种因素似乎有助于T细胞功能障碍，但HCV颗粒在此过程中的作用尚未确定。在这里，我们表明，原代人CD4+和CD8 + T细胞与含HCV RNA的血清，含HCV-RNA的细胞外囊泡（EV），细胞培养衍生的HCV颗粒（HCVpp）和HCV包膜假型逆转录病毒颗粒（HCVpp）的孵育抑制TCR介导的信号传导。由于HCVpp仅含有E1和E2，因此我们检测了HCV E2对TCR信号通路的影响。HCV E2表达再现了HCV颗粒诱导的TCR抑制。高度保守的51个核苷酸（nt）RNA序列足以抑制TCR信号传导。表达HCV E2编码RNA的细胞含有预测为Dicer底物的短病毒衍生RNA，其靶向参与Src激酶信号传导的磷酸酶（PTPRE）。含有HCV E2 RNA的T细胞和肝细胞的PTPRE蛋白水平降低。6nt的突变消除了预测的Dicer相互作用，并恢复了PTPRE表达和近端TCR信号传导。HCV RNA不抑制PMA和Ionomycin诱导的远端TCR信号传导;然而，HCV E2蛋白抑制远端TCR信号传导。这种抑制需要淋巴细胞特异性酪氨酸激酶（Lck）。Lck磷酸化HCV E2的一个保守的酪氨酸（Y613），磷酸化E2抑制NFAT的核转位。Y613的突变恢复了远端TCR信号传导，即使在HCVpps的背景下。因此，HCV颗粒将病毒RNA和E2蛋白递送至T细胞，并且这些分别抑制近端和远端TCR信号传导。HCV颗粒的这些作用可能有助于建立感染并有助于病毒持续存在。在全球范围内，约有2亿人持续感染丙型肝炎病毒（HCV）。HCV建立持续感染的机制是复杂的，并且几种宿主和病毒因素似乎有助于HCV逃避免疫清除的能力。通过T细胞受体（TCR）的T细胞活化是产生适应性免疫应答的重要第一步。虽然HCV感染与T细胞功能受损有关，但这种功能障碍的机制仍知之甚少。在这里，我们证明了HCV颗粒通过干扰由TCR激活触发的近端和远端信号来抑制T细胞活化。首先，将HCV包膜（E2）RNA加工成靶向调节磷酸酶的小RNA，抑制近端TCR信号传导。第二，淋巴细胞特异性Src激酶（Lck）在酪氨酸613（Y613）处磷酸化HCV E2，并且磷酸-E2抑制活化的NFAT的核转位，减少远端TCR活化信号。所涉及的RNA和蛋白质基序在所有HCV分离株中高度保守，并且突变恢复TCR信号传导。因此，HCV颗粒通过两种不同的机制干扰TCR信号传导并损害T细胞活化。这可能有助于HCV感染期间的HCV持久性和T细胞功能障碍。
T cell receptor (TCR) signaling is required for T-cell activation, proliferation, differentiation, and effector function. Hepatitis C virus (HCV) infection is associated with impaired T-cell function leading to persistent viremia, delayed and inconsistent antibody responses, and mild immune dysfunction. Although multiple factors appear to contribute to T-cell dysfunction, a role for HCV particles in this process has not been identified. Here, we show that incubation of primary human CD4+ and CD8+ T-cells with HCV RNA-containing serum, HCV-RNA containing extracellular vesicles (EVs), cell culture derived HCV particles (HCVcc) and HCV envelope pseudotyped retrovirus particles (HCVpp) inhibited TCR-mediated signaling. Since HCVpp’s contain only E1 and E2, we examined the effect of HCV E2 on TCR signaling pathways. HCV E2 expression recapitulated HCV particle-induced TCR inhibition. A highly conserved, 51 nucleotide (nt) RNA sequence was sufficient to inhibit TCR signaling. Cells expressing the HCV E2 coding RNA contained a short, virus-derived RNA predicted to be a Dicer substrate, which targeted a phosphatase involved in Src-kinase signaling (PTPRE). T-cells and hepatocytes containing HCV E2 RNA had reduced PTPRE protein levels. Mutation of 6 nts abolished the predicted Dicer interactions and restored PTPRE expression and proximal TCR signaling. HCV RNA did not inhibit distal TCR signaling induced by PMA and Ionomycin; however, HCV E2 protein inhibited distal TCR signaling. This inhibition required lymphocyte-specific tyrosine kinase (Lck). Lck phosphorylated HCV E2 at a conserved tyrosine (Y613), and phospho-E2 inhibited nuclear translocation of NFAT. Mutation of Y613 restored distal TCR signaling, even in the context of HCVpps. Thus, HCV particles delivered viral RNA and E2 protein to T-cells, and these inhibited proximal and distal TCR signaling respectively. These effects of HCV particles likely aid in establishing infection and contribute to viral persistence. Globally, approximately 200 million people are persistently infected with Hepatitis C virus (HCV). Mechanisms by which HCV establishes persistent infection are complex, and several host and viral factors appear to contribute to the ability of HCV to evade immune clearance. T cell activation through the T cell receptor (TCR) is an essential first step in the generation of an adaptive immune response. Although HCV infection is associated with impaired T cell function, the mechanisms for this dysfunction are poorly understood. Here, we demonstrate that HCV particles inhibit T cell activation by interfering with proximal and distal signals that are triggered by activation through the TCR. First, HCV envelope (E2) RNA was processed into a small RNA that targeted a regulatory phosphatase, inhibiting proximal TCR signaling. Second, the lymphocyte specific Src kinase (Lck) phosphorylated HCV E2 at tyrosine 613 (Y613), and phospho-E2 inhibited nuclear translocation of activated NFAT, reducing distal TCR activation signals. The RNA and protein motifs involved are highly conserved among all HCV isolates, and mutation restored TCR signaling. Thus, HCV particles interfere with TCR signaling and impair T cell activation using two distinct mechanisms. This may contribute to HCV persistence and T cell dysfunction during HCV infection.