Supplementary Material for: Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent

<b><i>Background:</i></b> In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. <b><i>Methods:</i></b> All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. <b><i>Results:</i></b> The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. <b><i>Conclusion:</i></b> These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.

<b><i>研究背景：</i></b> 免疫功能低下患者中，人类巨细胞病毒（human cytomegalovirus, HCMV）感染是引发发病率升高与死亡率增加的主要原因。细胞因子信号转导抑制因子（Suppressor of cytokine signaling, SOCS）家族蛋白是贾纳斯激酶/信号转导与转录激活因子（janus kinase/signal transducer and activator of transcription, JAK/STAT）通路的强效负调控因子。本研究推测人类巨细胞病毒可利用SOCS1和/或SOCS3以实现自身增殖优势。
<b><i>实验方法：</i></b> 所有实验均采用原代人肺微血管内皮细胞（primary human lung-derived microvascular endothelial cells, HMVEC）开展。通过向细胞中转染小干扰RNA（small interfering RNA, siRNA）沉默SOCS1与SOCS3的表达。人类巨细胞病毒在人肺成纤维细胞MRC5中扩增并滴定其病毒滴度。分别采用实时荧光定量聚合酶链式反应（real-time PCR）与蛋白质免疫印迹（Western blot）检测mRNA与蛋白质的表达水平。
<b><i>实验结果：</i></b> 本研究数据显示，人类巨细胞病毒在原代人肺微血管内皮细胞中的高效增殖依赖于SOCS3蛋白。时间进程分析结果表明，感染细胞内的SOCS3蛋白水平呈升高趋势。靶向SOCS3的小干扰RNA（siSOCS3）转染可抑制病毒即刻早期、早期及晚期抗原的表达。与之一致的是，与转染阴性对照小干扰RNA（siCNTRs）的对照组相比，siSOCS3转染组培养体系中的人类巨细胞病毒滴度显著降低。与siCNTR处理的对照组相比，siSOCS3转染的感染细胞中STAT1与STAT2的磷酸化水平显著升高。
<b><i>研究结论：</i></b> 上述研究结果表明，SOCS3参与了人类巨细胞病毒介导的细胞免疫应答调控机制。