Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois, USA.
Virus and Prion Research Unit, USDA-ARS-National Animal Disease Center, Ames, Iowa, USA.
J Virol. 2019 Apr 3;93(8). doi: 10.1128/JVI.02000-18. Print 2019 Apr 15.
Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses. Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.
鉴定先天免疫的病毒拮抗剂，并确定它们是否有助于发病机制，对于开发控制新兴病毒的有效策略至关重要。以前，我们报道了由鼠冠状病毒编码的内切核酸酶（EndoU）在巨噬细胞中逃避宿主先天免疫防御方面发挥关键作用。在这里，我们询问了引起猪急性腹泻的猪流行性腹泻冠状病毒（PEDV）的 EndoU 活性是否在拮抗猪上皮细胞和巨噬细胞（病毒复制的部位）中的先天反应中起作用。我们通过将 EndoU 的催化组氨酸残基的密码子改变为丙氨酸（His226Ala），构建了 PEDV-Colorado 株（icPEDV-wt）和 EndoU 突变 PEDV（icPEDV-EnUmt）的感染性克隆。我们发现，icPEDV-wt 和 icPEDV-EnUmt 在干扰素（IFN）缺陷的 Vero 细胞中均能有效繁殖。相比之下，icPEDV-EnUmt 在猪上皮细胞（LLC-PK1）中的繁殖受到损害，我们在其中检测到 I 型和 III 型 IFNs 的早期和强烈的转录激活。用亲本科罗拉多株、icPEDV-wt 或 icPEDV-EnUmt 感染仔猪表明，所有病毒均在肠道中复制并引起腹泻；然而，icPEDV-EnUmt 感染动物的病毒脱落和死亡率降低。这些结果表明，EndoU 活性对于 PEDV 在永生化、IFN 缺陷的 Vero 细胞中的复制不是必需的，但对于抑制上皮细胞和巨噬细胞中的 IFN 反应很重要，这有助于复制、脱落和发病机理。我们得出结论，PEDV EndoU 活性是一种关键的毒力因子，可抑制 I 型和 III 型 IFN 反应。冠状病毒（CoVs）可以从动物库中出现到一个天真的宿主物种，导致具有高死亡率的大流行呼吸道或胃肠道疾病在人类或家畜。猪流行性腹泻病毒（PEDV），一种甲型冠状病毒（alpha-CoV），感染肠道上皮细胞和巨噬细胞，引起腹泻，并导致仔猪高死亡率。PEDV 如何抑制先天免疫反应尚不清楚。我们发现，突变一种病毒内切核酸酶，EndoU，导致一种病毒在巨噬细胞和上皮细胞中既激活 I 型干扰素反应又激活 III 型干扰素反应。这种干扰素的激活导致上皮细胞培养物中病毒复制受限，并与仔猪中病毒脱落和死亡率降低有关。这项研究揭示了 EndoU 活性作为 PEDV 感染毒力因子的作用，并为新兴冠状病毒提供了一种生成活疫苗候选物的方法。
