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

Date Published: January 12, 2017

Publisher: Public Library of Science

Author(s): Huichen Guo, Mei Huang, Quan Yuan, Yanquan Wei, Yuan Gao, Lejiao Mao, Lingjun Gu, Yong Wah Tan, Yanxin Zhong, Dingxiang Liu, Shiqi Sun, Yongchang Cao.

http://doi.org/10.1371/journal.pone.0170123

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.

Partial Text

Avian infectious bronchitis virus (IBV) is a member of the Coronaviridae family, which includes many human and animal pathogens of global concern, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome virus (MERS-CoV), and mouse hepatitis virus(MHV)[1]. IBV has a major economic impact on the global poultry industry because this prototype coronavirus considerably decreases hen egg production by impairing the upper respiratory and reproductive tracts of chickens [2]. IBV is an enveloped positive-stranded RNA virus with a 27–30 kb genome that encodes several polyproteins. The polyproteins of IBV are cleaved by viral proteases into at least 15 nonstructural proteins (NSPs) [3]. Four structural proteins, namely, spike protein (S), nucleocapsid protein (N), membrane protein (M), and small envelope protein (E), are encoded by subgenomic RNA species [4]. The binding and entry of IBV into host cells require interactions between cellular surface receptors and viral structural proteins that are involved in the attachment stage. Nearly all IBV field isolates can only be propagated in embryonated chicken eggs or transiently proliferated in primary chicken embryo kidney cells. However, the Beaudette strain, a cell-adapted strain, replicates efficiently in various cultured mammalian cell lines, including African green monkey kidney cells (Vero), human liver cancer cells, lung cancer cell lines, and baby hamster kidney cells through serial passages [5–8]. However, the detailed mechanism underlying virus-host interactions during viral attachment and entry remains uninvestigated. Here, we used the Beaudette strain as an in vitro model to study the mechanism of IBV infection.

Viral infection comprises several steps, and viral attachment to host cells is the first step that determines tissue tropism, host specificity, and viral pathogenicity [34]. Although some viruses can recognize numerous host species, other viruses have limited host ranges because of their requirement for specific host cell receptors. In addition to special receptors, lipid rafts, as membrane components that ubiquitously exist in all cell types [35], are also involved in the regulation of endocytosis, intracellular trafficking of growth factor receptors, and localization of endosomes in cells [36]. A growing number of studies in the past two decades have reported that lipid rafts are crucial in the pathogenesis of many viruses [37,38] and bacteria [39]. Moreover, lipid rafts are involved in different stages of the viral life cycle, such as viral entry and release.

 

Source:

http://doi.org/10.1371/journal.pone.0170123