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.

胆固醇25-羟化酶（CH25H）最近已被确定为宿主限制性因子，可通过催化25-羟胆固醇（25HC）的产生发挥抗病毒作用。感染某些病毒后，可以迅速诱导CH25H。自1980年代末发现以来，猪生殖和呼吸综合症病毒（PRRSV）是一种动脉病毒，已被列为最重要的猪病原体之一。在这项研究中，我们发现PRRSV感染通过迄今未知的机制显着下调了细胞中CH25H的表达，这表明CH25H发挥了针对PRRSV的抗病毒活性。确实，CH25H的过表达抑制了PRRSV的复制，而短干扰RNA（siRNA）导致的CH25H的敲低促进了PRRSV的感染。25HC的抗PRRSV效应通过抑制病毒渗透来发挥作用。有趣的是，缺乏羟化酶活性的CH25H突变体（CH25H-M）仍然抑制PRRSV感染。使用酵母双杂交系统进行筛选，然后进行共免疫沉淀和免疫荧光共定位分析，确认CH25H和CH25H-M均与PRRSV的非结构蛋白1α（nsp1α）相互作用。出乎意料的是，与CH25H或CH25H-M共表达后，nsp1α的表达下降。详细的分析表明，CH25H / CH25H-M可以通过泛素-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，我们的发现表明，CH25H通过靶向病毒穿透以及降解nsp1α来限制PRRSV复制，从而揭示了CH25H使用的新型抗病毒机制。使用酵母双杂交系统进行筛选，然后进行共免疫沉淀和免疫荧光共定位分析，确认CH25H和CH25H-M均与PRRSV的非结构蛋白1α（nsp1α）相互作用。出乎意料的是，与CH25H或CH25H-M共表达后，nsp1α的表达下降。详细的分析表明，CH25H / CH25H-M可以通过泛素-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，我们的发现表明，CH25H通过靶向病毒穿透以及降解nsp1α来限制PRRSV复制，从而揭示了CH25H使用的新型抗病毒机制。使用酵母双杂交系统进行筛选，然后进行共免疫沉淀和免疫荧光共定位分析，确认CH25H和CH25H-M均与PRRSV的非结构蛋白1α（nsp1α）相互作用。出乎意料的是，与CH25H或CH25H-M共表达后，nsp1α的表达下降。详细分析表明，CH25H / CH25H-M可以通过泛素-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，我们的发现表明，CH25H通过靶向病毒穿透以及降解nsp1α来限制PRRSV复制，从而揭示了CH25H使用的新型抗病毒机制。详细的分析表明，CH25H / CH25H-M可以通过泛素-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，我们的发现表明，CH25H通过靶向病毒穿透以及降解nsp1α来限制PRRSV复制，从而揭示了CH25H使用的新型抗病毒机制。详细的分析表明，CH25H / CH25H-M可以通过泛素-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，我们的发现表明，CH25H通过靶向病毒穿透以及降解nsp1α来限制PRRSV复制，从而揭示了CH25H使用的新型抗病毒机制。

重要事项PRRSV一直是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。已发现CH25H具有广泛的抗病毒作用；因此，它是开发抗PRRSV药物的有吸引力的目标。在这里，我们证明CH25H是在猪肺泡巨噬细胞中高表达的干扰素刺激基因。CH25H不仅通过产生25HC来抑制病毒渗透，而且通过遍在蛋白-蛋白酶体途径降解病毒蛋白来发挥其抗PRRSV的作用，这表明CH25H是开发抗病毒疗法的候选药物。但是，PRRSV感染似乎会主动降低CH25H表达以促进病毒复制，从而突出PRRSV及其宿主之间的复杂博弈。