Li Ling, Zhuang Pingping, Cheng Ziqiang, Yang Jie, Bi Jianmin, Wang Guihua
Department of Fundamental Veterinary, College of Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China.
China Animal Husbandry Industry Co. Ltd., Beijing 10070, China.
J Vet Sci. 2020 May;21(3):e49. doi: 10.4142/jvs.2020.21.e49.
Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components.
The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV.
Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction.
The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells.
Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.
鸡群中禽白血病病毒J亚群（ALV-J）与网状内皮组织增殖病病毒（REV）共感染很常见，且共感染协同致病作用的分子机制尚不清楚。外泌体已被确定为逆转录病毒发病机制中的新角色。外泌体的不同功能取决于其携带成分。
本研究旨在探讨外泌体中共同调控的差异表达蛋白对ALV-J和REV共感染的作用。
通过免疫荧光显微镜检测感染ALV-J、REV或两者的鸡胚成纤维细胞（CEF）中的病毒复制。然后，我们通过质谱分析从单独感染以及同时感染ALV-J和REV的鸡胚成纤维细胞（CEF）上清液中分离出的外泌体。KEGG通路富集分析外泌体中共同调控的差异表达蛋白。接下来，我们沉默并过表达含三重基序蛋白62（TRIM62），以评估TRIM62对病毒复制的影响，并通过定量逆转录聚合酶链反应测定NCK关联蛋白1（NCKAP1）和肌动蛋白相关蛋白2/3复合体亚基5（ARPC5）的表达水平。
结果表明，ALV-J和REV共感染促进彼此的复制。鉴定出30种蛋白质，包括TRIM62、NCK关联蛋白1（NCKAP1，也称为Nap125）和Arp2/3-5、ARPC5。NCKAP1和ARPC5参与肌动蛋白细胞骨架途径。TRIM62负调控病毒复制，在与TRIM62共感染的CEF细胞中，对REV的抑制作用比对ALV-J更显著。此外，TRIM62降低了共感染CEF细胞中NCKAP1的表达，并增加了ARPC5的表达。
总体而言，我们的结果表明，ALV-J和REV共感染相互竞争促进彼此的复制，肌动蛋白细胞骨架在共感染机制中起重要作用，且TRIM62调控肌动蛋白细胞骨架。
