Wang Xiaoyan, Shen Huanjun, Du Hong, Jiang Hong, Wang Pingzhong, Zhang Ying
Department of Infectious Diseases, Tangdu Hospital，The Fourth Military University, Xi'an 710038, PR China.
J Med Microbiol. 2025 Jun;74(6). doi: 10.1099/jmm.0.001989.
Hantaan virus (HTNV) predominantly infects human vascular endothelial cells (ECs) and causes increased vascular permeability, triggering haemorrhagic fever with renal syndrome, mainly in Asia. Previous studies have shown that endothelial permeability is regulated in part by the break of cell-cell adherens junctions (AJs). However, the intracellular mechanisms by which HTNV induces EC hyperpermeability via AJs remain unclear. We hypothesize that HTNV activates TLR4, and its downstream TRAF6 interacts with SFK, leading to the phosphorylation of adhesion junction-associated proteins and increased cell permeability. The present study aimed to investigate the molecular mechanism by which Src family kinases (SFKs) modulate AJs and affect permeability. Real-time PCR (RT-PCR) and Western blot were used to assess TLR4, TRAF6 and SFK expression; Western blot was used to analyse the protein expression of AJs; small interfering RNAs (siRNAs) were used to inhibit gene expression in the human umbilical vein endothelial cells (HUVECs) and the distribution of vascular endothelial cadherin (VE-cadherin) was observed by immunofluorescence. HUVECs infected by HTNV displayed a lower permeability after a siRNA knockdown of TLR4 (si-TLR4). Moreover, HTNV increased the expression of TRAF6 and the phosphorylation of Src and AJs. After siRNA knockdown of TRAF6 (si-TRAF6), a decrease in the phosphorylation of Src and VE-cadherin was observed in HTNV-infected ECs compared to that in siRNA controls. These data, for the first time, indicated that HTNV-induced upregulation of AJ phosphorylation is regulated by the TLR4/TRAF6/SFK signalling pathway.
汉坦病毒（HTNV）主要感染人类血管内皮细胞（ECs），导致血管通透性增加，引发肾综合征出血热，主要发生在亚洲。先前的研究表明，内皮通透性部分受细胞间黏附连接（AJs）的破坏调节。然而，HTNV通过AJs诱导EC高通透性的细胞内机制仍不清楚。我们假设HTNV激活Toll样受体4（TLR4），其下游的肿瘤坏死因子受体相关因子6（TRAF6）与Src家族激酶（SFK）相互作用，导致黏附连接相关蛋白磷酸化并增加细胞通透性。本研究旨在探讨SFK调节AJs并影响通透性的分子机制。采用实时定量聚合酶链反应（RT-PCR）和蛋白质免疫印迹法评估TLR4、TRAF6和SFK的表达；用蛋白质免疫印迹法分析AJs的蛋白表达；使用小干扰RNA（siRNAs）抑制人脐静脉内皮细胞（HUVECs）中的基因表达，并通过免疫荧光观察血管内皮钙黏蛋白（VE-cadherin）的分布。用siRNA敲低TLR4（si-TLR4）后，HTNV感染的HUVECs通透性降低。此外，HTNV增加了TRAF6的表达以及Src和AJs的磷酸化。与siRNA对照组相比，用siRNA敲低TRAF6（si-TRAF6）后，HTNV感染的ECs中Src和VE-cadherin的磷酸化减少。这些数据首次表明，HTNV诱导的AJ磷酸化上调受TLR4/TRAF6/SFK信号通路调控。
