The Important Role of Lipid Raft-Mediated Attachment in the Infection of Cultured Cells by Coronavirus Infectious Bronchitis Virus Beaudette Strain

Guo, Huichen; Huang, Mei; Yuan, Quan; Wei, Yanquan; Gao, Yuan; Mao, Lejiao; Gu, Lingjun; Tan, Yong Wah; Zhong, Yanxin; Liu, Dingxiang; Sun, Shiqi

Lipid rafts are required for IBV infection, but are not involved in viral genome replication and particle release. To further study the participation of lipid rafts in the early stages of viral infection, as well as to identify the specific involvement of lipid rafts in viral endocytosis, lipid rafts were inactivated with MβCD. To determine the role of lipid rafts in IBV attachment, host cells were pre-treated with MβCD for 1 h at 4˚C, incubated with IBV for 1 h at 4˚C, and then incubated at 37˚C for 2, 4, 6, and 8 h. To determine the role of lipid rafts in IBV entry, host cells were pre-incubated with IBV for 1 h at 4˚C, treated with MβCD for 1 h at 4˚C, and then cultured at 37˚C for 2, 4, 6, and 8 h. Vero cells without the MβCD treatment were set as the control. To assess viral replication, IBV N protein was subjected to Western blot analysis. As shown in Fig 5A, IBV N was detectable in the control group with increasing hpi. MβCD treatment decreased IBV N levels compared with that of the control group. In particular, MβCD treatment prior to IBV attachment significantly repressed IBV N expression. This result suggested that MβCD treatment blocked viral particles from binding with the cell membrane. To further confirm the function of lipid rafts in IBV endocytosis, the IBV-susceptible cell lines A549 and DF1 were treated with MβCD before or after IBV attachment (Fig 5B and 5C ). The depletion of lipid rafts by MβCD suppressed IBV N expression in both cell lines when cells were treated with MβCD before IBV attachment. However, this result was not observed when cells received MβCD treatment after IBV attachment. These results indicated that MβCD treatment directly inhibited IBV attachment and that drug-induced lipid raft disruption mainly blocked IBV attachment to host cells. Therefore, lipid rafts are crucial to virus endocytosis and are required for IBV attachment.