Li Xin, Feng Yiyi, Liu Weiwei, Tan Lei, Sun Yingjie, Song Cuiping, Liao Ying, Xu Chenggang, Ren Tao, Ding Chan, Qiu Xusheng
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Front Microbiol. 2022 May 11;13:874331. doi: 10.3389/fmicb.2022.874331. eCollection 2022.
Virus infection can lead to the production of interferon, which activates the JAK/STAT pathway and induces the expression of multiple downstream interferon-stimulated genes (ISGs) to achieve their antiviral function. Cytidine/uridine monophosphate kinase 2 () gene has been identified as an ISG in human and fish, and is also known as a rate-limiting enzyme in mitochondria to maintain intracellular UTP/CTP levels, which is necessary for mitochondrial DNA synthesis. By mining previous microarray data, it was found that both Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV) infection can lead to the significant upregulation of chicken gene. However, little is known about the function of gene in chickens. In the present study, the open reading frame (ORF) of chicken CMPK2 (chCMPK2) was cloned from DF-1, a chicken embryo fibroblasts cell line, and subjected to further analysis. Sequence analysis showed that chCMPK2 shared high similarity in amino acid with CMPK2 sequences from all the other species, especially reptiles. A thymidylate kinase (TMK) domain was identified in the C-terminus of chCMPK2, which is highly conserved among all species. , AIV infection induced significant increases in chCMPK2 expression in DF-1, HD11, and the chicken embryonic fibroblasts (CEF), while obvious increase only detected in DF-1 cells and CEF cells after NDV infection. , the expression levels of were up-regulated in several tissues from AIV infected chickens, especially the brain, spleen, bursa, kidney, intestine, heart and thymus, and notable increase of was detected in the bursa, kidney, duodenum, lung, heart, and thymus during NDV infection. Here, using MDA5 and IFN-β knockdown cells, we demonstrated that as a novel ISG, chCMPK2 could be regulated by the MDA5/IFN-β pathway. The high expression level of exogenous chCMPK2 displayed inhibitory effects on AIV and NDV as well as reduced viral RNA in infected cells. We further demonstrated that Asp135, a key site on the TMK catalytic domain, was identified as critical for the antiviral activities of chCMPK2. Taken together, these data demonstrated that chCMPK2 is involved in the chicken immune system and may play important roles in host anti-viral responses.
病毒感染可导致干扰素的产生，干扰素激活JAK/STAT信号通路并诱导多个下游干扰素刺激基因（ISGs）的表达，以实现其抗病毒功能。胞苷/尿苷单磷酸激酶2（）基因已被鉴定为人类和鱼类中的一种ISG，并且还被认为是线粒体中维持细胞内UTP/CTP水平的限速酶，这对于线粒体DNA合成是必需的。通过挖掘先前的微阵列数据，发现禽流感病毒（AIV）和新城疫病毒（NDV）感染均可导致鸡基因的显著上调。然而，关于鸡基因的功能知之甚少。在本研究中，从鸡胚成纤维细胞系DF-1中克隆了鸡CMPK2（chCMPK2）的开放阅读框（ORF），并进行了进一步分析。序列分析表明，chCMPK2与所有其他物种（尤其是爬行动物）的CMPK2序列在氨基酸上具有高度相似性。在chCMPK其二的C末端鉴定出一个胸苷酸激酶（TMK）结构域，该结构域在所有物种中高度保守。，AIV感染诱导DF-1、HD11和鸡胚成纤维细胞（CEF）中chCMPK2表达显著增加，而NDV感染后仅在DF-1细胞和CEF细胞中检测到明显增加。，在AIV感染鸡的多个组织中，尤其是脑、脾、法氏囊、肾、肠、心脏和胸腺中，的表达水平上调，在NDV感染期间，在法氏囊、肾、十二指肠、肺、心脏和胸腺中检测到显著增加。在此，我们使用MDA5和IFN-β敲低细胞证明，作为一种新型ISG，chCMPK2可受MDA5/IFN-β信号通路调控。外源性chCMPK2的高表达水平对AIV和NDV具有抑制作用，并降低了感染细胞中的病毒RNA。我们进一步证明，TMK催化结构域上关键位点Asp135对于chCMPK2的抗病毒活性至关重要。综上所述，这些数据表明chCMPK2参与鸡的免疫系统，并可能在宿主抗病毒反应中发挥重要作用。
