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

Primer sequences S1 forward 5 0 -TGAGATTGAAAGCAACGCCAGTTG-3 0 S1 reverse 5 0 -CTTACCATAACTAACATAAGGGC-3 0 GAPDH forward 5 0 -CGGCATCCACGAAACTAC-3 0 GAPDH reverse To directly observe the dynamic changes of co-localization and to verify the results of the flotation gradients, IFA was performed to investigate the interaction between IBV E protein and lipid rafts via confocal microscopy. Vero cells overexpressing IBV E protein were harvested for IFA detection at 8, 16, and 24 h post-transfection. IFA staining was performed with anti-IBV E and FITC-conjugated GM1 antibodies. As shown in Fig 2B, IBV E protein appeared both in the plasma membrane and cytoplasm at 8 h post-transfection and then gradually translocated to the plasma membrane. At 24 h post-transfection, IBV E protein was mainly distributed in the plasma membrane with GM1. The transfer between the intracellular and plasma membrane indicated that IBV E protein is functionally related to lipid rafts in a time-dependent manner after transfection. The results suggested that lipid rafts may be required for some stages of IBV infection.