- 作者: Xiaoling Yuan, Tao Hu, Hanwen He, Huan Qiu, Xuan Wu, Jingxian Chen, Minmin Wang, Cheng Chen and Shenghai Huang
- 作者服務機構: 1. Department of Microbiology, School of Basic Medicine, Anhui Medical University, Hefei, Anhui Province 230032, People’s Republic of China 2. School of Life Sciences, Anhui Medical University, Hefei, Anhui Province 230032, People’s Republic of China
- 中文摘要:
- 英文摘要:
Background
Respiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated.
Methods
We compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry.
Results
The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection.
Conclusion
The RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin. - 中文關鍵字:
- 英文關鍵字: Respiratory syncytial virus (RSV), Toll-like receptor 4 (TLR4), Nucleolin (C23), Virus infection, N2a cells