The Human Cytomegalovirus US27 Gene Product Constitutively Activates Antioxidant Response Element-Mediated Transcription through Gβγ, Phosphoinositide 3-Kinase, and Nuclear Respiratory Factor 1
The Human Cytomegalovirus US27 Gene Product Constitutively Activates Antioxidant Response Element-Mediated Transcription through Gβγ, Phosphoinositide 3-Kinase, and Nuclear Respiratory Factor 1
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DOI：
10.1128/jvi.00644-18
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发表时间：
2018-12-01
影响因子：
5.4
通讯作者：
Spencer, Juliet, V
中科院分区：
文献类型：
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作者：
Boeck, Jordan M.;Stowell, Gregory A.;Spencer, Juliet, V
人巨细胞病毒（HCMV）是一种广泛存在的病原体，在宿主持续感染期间调节宿主趋化因子信号。HCMV编码四种与G蛋白偶联受体（gpcr）趋化因子受体家族同源的蛋白：US27、US28、UL33和UL78。四种受体中的每一种都调节宿主CXCR4信号传导。US28、UL33和UL78损害CXCR4信号转导结果，而US27增强信号转导结果，这可以通过增加钙动员和细胞向CXCL12的迁移来证明。为了研究US27在病毒感染期间对CXCR4的影响，将成纤维细胞感染bac衍生的HCMV临床菌株TB40/E-mCherry （WT）或缺乏US27的突变体（TB40/E-mCherry-US27 Delta）或所有四种gpcr (TB40 E-mCherry-all Delta)，或仅表达US27，但不表达US28、UL33或UL78 （TB40/E-mCherry- us27wt）。在WT和us27wt感染的成纤维细胞中，CXCR4基因表达显著升高。这种效应在感染后3小时明显，表明来自亲本病毒粒子的US27增强了CXCR4的表达。报告基因分析显示US27增加了抗氧化反应元件（ARE）调控的转录活性，siRNA处理表明这种作用是由NRF-1介导的，NRF-1是CXCR4的主要转录因子。免疫荧光显微镜证实，与模拟或US27 delta感染细胞相比，wt感染细胞的NRF-1易位增加。靶向G(β) γ和磷酸肌肽3激酶（PI3K）的化学抑制剂抑制了are驱动转录的增加，暗示这些蛋白是us27刺激基因转录的介质。这项工作确定了HCMV US27激活的第一个信号通路，并可能揭示这种孤儿病毒受体在感染期间刺激应激反应基因的新调控功能。人类巨细胞病毒（HCMV）是世界上最常见的先天性感染，可导致耳聋、失明和其他严重的出生缺陷。CXCR4是一种人类趋化因子受体，对胎儿发育和免疫反应都至关重要。我们发现HCMV蛋白US27通过激活转录因子核呼吸因子1 （NRF-1）刺激CXCR4的表达增加。NRF-1调节含有抗氧化反应元件（ARE）的应激反应基因，当US27存在时，HCMV感染与许多应激反应基因的表达增加有关。我们的研究结果表明，US27蛋白激活NRF-1/ARE通路，刺激CXCR4和其他应激反应基因的高表达，这可能有利于病毒复制和/或免疫逃避。
Human cytomegalovirus (HCMV) is a widespread pathogen that modulates host chemokine signaling during persistent infection in the host. HCMV encodes four proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. Each of the four receptors modulates host CXCR4 signaling. US28, UL33 and UL78 impair CXCR4 signaling outcomes while US27 enhances signaling, as evidenced by increased calcium mobilization and cell migration to CXCL12. To investigate effects of US27 on CXCR4 during virus infection, fibroblasts were infected with BAC-derived clinical strain HCMV TB40/E-mCherry (WT) or mutants lacking US27 (TB40/E-mCherry-US27 Delta) or all four GPCRs (TB40 E-mCherry-all Delta), or expressing only US27, but not US28, UL33, or UL78 (TB40/E-mCherry-US27wt). CXCR4 gene expression was significantly higher in WT and US27wt-infected fibroblasts. This effect was evident at 3 hours post-infection, suggesting that US27 derived from the parental virion enhanced CXCR4 expression. Reporter gene assays demonstrated US27 increased transcriptional activity regulated by the antioxidant response element (ARE), and siRNA treatment indicated this effect was mediated by NRF-1, the primary transcription factor for CXCR4. Increased translocation of NRF-1 into the nucleus of WT-infected cells compared to mock- or US27 Delta-infected cells was confirmed by immunofluorescence microscopy. Chemical inhibitors targeting G(beta)gamma and phosphoinositide 3-kinase (PI3K) ablated the increase in ARE-driven transcription, implicating these proteins as mediators of US27-stimulated gene transcription. This work identifies the first signaling pathway activated by HCMV US27 and may reveal a novel regulatory function for this orphan viral receptor in stimulating stress response genes during infection.IMPORTANCE Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, causing deafness, blindness, and other serious birth defects. CXCR4 is a human chemokine receptor that is crucial for both fetal development and immune responses. We found that the HCMV protein US27 stimulates increased expression of CXCR4 through activation of the transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 regulates stress response genes that contain the antioxidant response element (ARE), and HCMV infection is associated with increased expression of many stress response genes when US27 is present. Our results show that the US27 protein activates the NRF-1/ARE pathway, stimulating higher expression of CXCR4 and other stress response genes, which is likely to be beneficial for virus replication and/or immune evasion.