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

Abstract

Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-β-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage.

Fig 1. IBV structural proteins are associated with lipid rafts in IBV-infected Vero cells.

Vero cells infected with IBV at 1 MOI for 24 h were lysed with TNEV buffer containing 1% Brij. Lysates were then analyzed by sucrose gradient centrifugation. Samples from each fraction were evaluated via Western blot analysis with specific antibodies against E, N, S, NSP5, and NSP12. Both lipid raft and non-lipid raft fractions were detected with cav-1.

Fig 2. IBV E protein co-localizes with GM1.

Vero cells were transfected with plasmids expressing IBV structural protein E and collected in TNEV buffer with 1% Brij at 16 h and 24 h after transfection. Lysates were analyzed by sucrose gradient ultracentrifugation. (A) Samples from each fraction were evaluated through Western blot analysis with specific antibodies against IBV E (above). The floating bands from sucrose gradient ultracentrifugation were collected and assessed by dot blot analysis with GM1 antibody (below). (B) Vero cells were transfected with plasmids encoding IBV E protein and fixed with 4% PFA at 8, 16, and 24 h post-transfection. The cells were then permeabilized and immunolabeled with IBV E and GM1 antibodies. Slides were analyzed via confocal laser scanning microscopy.

Fig 3. IBV structural proteins in the supernatant are unaffected by MβCD or Mevastatin treatment.

Vero cells with or without drug treatment were infected with IBV at 1 MOI for 0, 8, 12, 18, and 24 h. Supernatants were directly collected and the cells were lysed for Western blot assay. The samples were evaluated with specific antibodies against the IBV N protein. Tubulin was used as loading control marker.

Fig 4. IBV genome replication is evaluated by RT-PCR assay after MβCD or mevastatin treatment.

Drug-treated Vero cells after IBV infection at 1 MOI for 1 h at 37°C were collected at 0 h, 8 h, 12 h, 18 h, and 24 h. Total RNA was extracted from the cells. Negative-strand gRNA and subgenomic mRNA were detected by semi-quantitative RT-PCR. Line charts represent the tendency on the basis of PCR results.

Fig 5. Lipid rafts are involved in virus attachment.

Vero, A549, and DF1 cells were treated with MβCD before or after IBV infection at 10 MOI. Cells were incubated at 37°C for indicated time points. The cells were then lysed and analyzed by Western blot with specific antibodies against IBV N protein. β-actin was used as the loading control.