College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin, China.
Front Immunol. 2022 May 26;13:827681. doi: 10.3389/fimmu.2022.827681. eCollection 2022.
Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity . However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat () genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.
新兴流感病毒对人类和动物的健康构成威胁。最近已确定家猫是人畜共患流感病毒的潜在来源。基于 RNA 聚合酶 I (PolI) 启动子的流感病毒小基因组复制系统是研究聚合酶活性最广泛使用的工具。它可以帮助确定影响流感病毒聚合酶活性的宿主因素或病毒蛋白。然而，迄今为止，尚未在猫细胞中研究流感病毒聚合酶活性。在本研究中，通过生物信息学策略在猫基因组中相邻上游 28S 和下游 18S rRNA 基因之间的基因间间隔区中鉴定了猫 RNA PolI 启动子。预测了猫 RNA PolI 启动子的转录起始位点。从 CRFK 细胞中克隆了猫 RNA PolI 启动子，双荧光素酶报告基因检测表明，大小为 250bp 的启动子含有具有足够 PolI 启动子活性的序列。然后建立了基于猫 RNA PolI 启动子的流感病毒小基因组复制系统。使用该系统，确定猫 RNA PolI 启动子在猫细胞中的转录活性明显高于人源和鸡源 RNA PolI 启动子，并且马源（H3N8）流感病毒的聚合酶活性高于人流感病毒（H1N1）和犬源（H3N2）流感病毒。此外，观察到猫抗黏液病毒蛋白 1（Mx1）和巴洛昔韦以剂量依赖性方式抑制流感病毒聚合酶活性。我们的研究将有助于进一步研究流感病毒在猫中宿主适应性和跨种传播的分子机制。
