猪三角洲冠状病毒(PDCoV)是在许多猪生产国发现的肠病原体。会引起急性腹泻,呕吐,脱水,和新生仔猪的死亡,严重影响生猪养殖业的发展。迄今为止,我们对PDCoV发病机制及其与宿主细胞因子相互作用的认识尚不完全.使用Co-IP与LC/MS-MS耦合,我们确定了在LLC-PK1细胞中可能与PDCoV相互作用的67种蛋白质;选择了5种鉴定的蛋白质进行进一步评估(IMMT,STAT1、XPO5、PIK3AP1和TMPRSS11E)。五个LLC-PK1细胞系,每个都有一个感兴趣的基因被击倒,使用CRISPR/cas9构建。在这些敲低细胞系中,仅STAT1KD导致显著更高的病毒产量。敲除了剩下的四个基因,在不同程度上,在较低的病毒产量野生型LLC-PK1细胞。STAT1的缺失没有显著影响PDCoV对细胞的附着,但确实导致病毒内化增加。此外,PDCoV感染刺激STAT1(IFIT1,IFIT2,RADS2,ISG15,MX1和OAS1)下游干扰素刺激基因(ISG)的表达,而STAT1的敲低导致所有6个ISG的表达降低80%以上。我们的发现表明STAT1与PDCoV相互作用,并在PDCoV感染中起负调控作用。
Porcine deltacoronavirus (PDCoV) is an enteropathogen found in many pig producing countries. It can cause acute diarrhea, vomiting, dehydration, and death in newborn piglets, seriously affecting the development of pig breeding industries. To date, our knowledge of the pathogenesis of PDCoV and its interactions with host cell factors remains incomplete. Using Co-IP coupled with LC/MS-MS, we identified 67 proteins that potentially interact with PDCoV in LLC-PK1 cells; five of the identified proteins were chosen for further evaluation (IMMT,
STAT1, XPO5, PIK3AP1, and TMPRSS11E). Five LLC-PK1 cell lines, each with one of the genes of interest knocked down, were constructed using CRISPR/cas9. In these knockdown cells lines, only STAT1KD resulted in a significantly greater virus yield. Knockdown of the remaining four genes resulted, to varying degrees, in a lower virus yield that wild-type LLC-PK1 cells. The absence of
STAT1 did not significantly affect the attachment of PDCoV to cells, but did result in increased viral internalization. Additionally, PDCoV infection stimulated expression of interferon stimulated genes (ISGs) downstream of
STAT1 (IFIT1, IFIT2, RADS2, ISG15, MX1, and OAS1) while knockdown of
STAT1 resulted in a greater than 80 % decrease in the expression of all six ISGs. Our findings show that
STAT1 interacts with PDCoV, and plays a negative regulatory role in PDCoV infection.