作为专性寄生虫,病毒已经进化出多种策略来逃避宿主的免疫防御。操纵宿主蛋白酶体系统以降解特定有害因素是常见的病毒对策。鉴定猪繁殖与呼吸综合征病毒(PRRSV)降解蛋白酶体的宿主蛋白,我们对蛋白酶体抑制剂MG132处理的PRRSV感染的Marc-145细胞进行了定量蛋白质组学筛选.数据显示,程序性细胞死亡4(PDCD4)的表达水平被PRRSV强烈下调,并被MG132显著挽救。进一步的研究证实,PRRSV感染诱导PDCD4从细胞核到细胞质的易位,病毒非结构蛋白9(Nsp9)通过激活Akt-mTOR-S6K1通路促进了PDCD4蛋白酶体在细胞质中的降解。Nsp9的C末端结构域负责PDCD4降解。至于PDCD4在PRRSV感染中的作用,我们证明了PDCD4敲低有利于病毒复制,虽然它的过表达显著减弱了复制,提示PDCD4是PRRSV的限制因子。机械上,我们发现PRRSV需要真核翻译起始因子4A(eIF4A)。PDCD4通过其两个MA3域内的四个位点(E249、D253、D414和D418)与eIF4A相互作用,在PRRSV的5'-非翻译区中破坏eIF4A介导的翻译起始,从而抑制PRRSV感染。一起,我们的研究揭示了PDCD4的抗病毒功能和拮抗PDCD4的病毒策略.这些结果将有助于我们对PRRSV采用的免疫逃避策略的理解,并为开发新的抗病毒靶标提供有价值的见解。猪繁殖与呼吸综合征病毒(PRRSV)感染给全球养猪业造成重大经济损失,难以有效控制。这里,使用定量蛋白质组学筛选,我们鉴定了程序性细胞死亡4(PDCD4)作为PRRSV蛋白酶体降解的宿主蛋白.我们证明了PDCD4通过与真核翻译起始因子4A相互作用来限制PRRSV的复制,这是病毒5'非翻译区中翻译起始所必需的。此外,PDCD4的两个MA3结构域内的四个位点被鉴定为负责其抗病毒功能。相反,PRRSV非结构蛋白9通过激活Akt-mTOR-S6K1通路促进PDCD4蛋白酶体在细胞质中的降解,从而削弱了抗PRRSV的功能。我们的工作揭示了PDCD4是PRRSV以前未被识别的宿主限制因子,并揭示了PRRSV开发了克服PDCD4的对策。这将为病毒-宿主相互作用和新的抗病毒靶标的开发提供新的见解。
As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted
PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for
PDCD4 degradation. As for the role of
PDCD4 during PRRSV infection, we demonstrated that
PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5\'-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of
PDCD4 and the viral strategy to antagonize
PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5\'-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome
PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.