Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms
Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms
复制标题
胆固醇 25-羟化酶通过酶活性依赖性和独立机制抑制猪繁殖和呼吸综合征病毒复制
DOI：
10.1128/jvi.00827-17
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发表时间：
2017-10-01
影响因子：
5.4
通讯作者：
Xiao, Shaobo
中科院分区：
文献类型：
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作者：
Ke, Wenting;Fang, Liurong;Xiao, Shaobo
摘要胆固醇25-羟基酶(CH25H)是新近发现的一种宿主限制因子，它通过催化25-羟基胆固醇(25HC)的产生而发挥抗病毒作用。CH25H可在感染某些病毒后迅速被诱导。猪繁殖与呼吸综合征病毒(PRRSV)是一种动脉病毒，自20世纪80年代末被发现以来，一直是猪最重要的病原体之一。在这项研究中，我们发现PRRSV感染显著下调细胞中CH25H的表达，这是一种迄今未知的机制，表明CH25H对PRRSV具有抗病毒活性。事实上，CH25H的过表达抑制了PRRSV的复制，而通过短干扰RNA(SiRNA)敲除CH25H促进了PRRSV的感染。25HC的抗PRRSV作用是通过抑制病毒穿透发挥作用的。有趣的是，一个缺乏羟基酶活性的CH25H突变体(CH25H-M)仍然抑制PRRSV的感染。用酵母双杂交系统筛选，免疫共沉淀和免疫荧光共定位分析证实CH25H和CH25H-M都与PRRSV的非结构蛋白1α(NSP1α)相互作用。与CH25H或CH25H-M共表达后，NSP1α的表达显著下降。详细分析表明，CH25H/CH25H-M可通过泛素-蛋白酶体途径降解NSP1α，NSP1α蛋白中的K169位点是泛素化的关键位点。综上所述，我们的研究结果表明，CH25H通过靶向病毒穿透和降解NSP1α来抑制PRRSV的复制，揭示了CH25H使用的一种新的抗病毒机制。PRRSV的重要性一直是对全球养猪业的持续威胁，目前的疫苗不足以提供可持续的控制。CH25H已被发现具有广泛的抗病毒作用，是抗PRRSV药物开发的一个有吸引力的靶点。在这里，我们证明了CH25H是一个干扰素刺激的基因，在猪肺泡巨噬细胞中高度表达。CH25H不仅通过产生25HC抑制病毒穿透，还通过泛素-蛋白酶体途径降解病毒蛋白发挥抗PRRSV作用，提示CH25H是抗病毒治疗药物的候选药物。然而，PRRSV感染似乎主动降低CH25H的表达以促进病毒复制，突显了PRRSV与其宿主之间的复杂博弈。
ABSTRACT Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so-far unknown mechanism, suggesting that CH25H exerts antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by short interfering RNA (siRNA) promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by coimmunoprecipitation and immunofluorescence colocalization analyses confirmed that both CH25H and CH25H-M interact with the nonstructural protein 1 alpha (nsp1α) of PRRSV. Unexpectedly, the expression of nsp1α decreased following coexpression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1α through the ubiquitin-proteasome pathway and that site K169 in the nsp1α protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration as well as degrading nsp1α, revealing a novel antiviral mechanism used by CH25H. IMPORTANCE PRRSV has been a continuous threat to the global swine industry, and current vaccines are insufficient to provide sustainable control. CH25H has been found to exert a broad antiviral effect; thus, it is an attractive target for the development of anti-PRRSV drugs. Here, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration but also by degrading viral protein through the ubiquitin-proteasome pathway, suggesting that CH25H is a candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.