Yang Saixia, Wang Yiwang, Yang Jifei, Tian Zhancheng, Wu Mengli, Sun Hualin, Zhang Xiaoqiang, Zhao Yaru, Luo Jianxun, Guan Guiquan, Yin Hong, Hao Rongzeng, Niu Qingli
State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
African Swine Fever Regional Laboratory of China (Lanzhou), Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou, Gansu, China.
Front Microbiol. 2024 Sep 24;15:1469166. doi: 10.3389/fmicb.2024.1469166. eCollection 2024.
ASFV C315R is homologous to the transcription factor TFIIB of large unclassified DNA viruses, and H359L is identical to the subunit 3 (RPB3) of eukaryotic RNA polymerase II. The C315R and H359L may play an important role in ASFV replication and transcription. Here, we evaluated the biological function of the and genes during virus replication and during infection in pigs. Results showed that C315R and H359L are highly conserved among ASFV genotype II strains; quantitative PCR (qPCR) and western blotting analyses revealed that and are early transcribed genes prior to viral DNA replication, but their protein expression is delayed. The immunofluorescence and western blotting analysis revealed that both proteins localized in the cell cytoplasm and nucleus at 24 h post infection, however, pH359L was mainly detected in the cell cytoplasm. Furthermore, overexpression of pH359L in MA104 cells significantly increased viral titer, RNA transcription levels, and viral protein expression levels, while overexpression of pC315R slightly enhanced ASFV replication. In contrast, siRNA targeting ASFV-H359L or C315R reduced replication efficiency in porcine macrophage culture compared to the parent ASFV-CN/SC/2019, demonstrating that and genes are necessary for ASFV replication. Finally, the functional role of C315R or H359L on PKR and eIF2α phosphorylation status and SG formation, as well as cytokine production were evaluated. These studies demonstrated that C315R and H359L are involved in virus replication processes in swine and play important roles in ASFV replication.
非洲猪瘟病毒C315R与大型未分类DNA病毒的转录因子TFIIB同源，而H359L与真核生物RNA聚合酶II的亚基3（RPB3）相同。C315R和H359L可能在非洲猪瘟病毒的复制和转录中发挥重要作用。在此，我们评估了 和 基因在病毒复制过程以及猪感染过程中的生物学功能。结果表明，C315R和H359L在非洲猪瘟病毒II型毒株中高度保守；定量PCR（qPCR）和蛋白质印迹分析显示， 和 是病毒DNA复制之前的早期转录基因，但其蛋白质表达延迟。免疫荧光和蛋白质印迹分析显示，感染后24小时，这两种蛋白质均定位于细胞质和细胞核中，然而，pH359L主要在细胞质中检测到。此外，在MA104细胞中过表达pH359L显著提高了病毒滴度、RNA转录水平和病毒蛋白表达水平，而pC315R的过表达则略微增强了非洲猪瘟病毒的复制。相比之下，与亲本非洲猪瘟病毒-CN/SC/2019相比，靶向非洲猪瘟病毒-H359L或C315R的小干扰RNA（siRNA）降低了猪巨噬细胞培养物中的复制效率，表明 和 基因是非洲猪瘟病毒复制所必需的。最后，评估了C315R或H359L对蛋白激酶R（PKR）和真核生物翻译起始因子2α（eIF2α）磷酸化状态、应激颗粒（SG）形成以及细胞因子产生的功能作用。这些研究表明，C315R和H359L参与猪体内的病毒复制过程，并在非洲猪瘟病毒复制中发挥重要作用。
