Lipid rafts are membrane microdomains enriched in sphingolipids and cholesterol. They contain lipids in liquid ordered phase and may correspond to
those membrane structures described as detergent-resistant membranes.
Apart from various cellular processes, lipid rafts have been reported to play a critical role in different aspects of the virus life cycle such as viral entry, protein transport and targeting, and assembly and budding (Nayak and Hui, 2004).
Cholesterol is a characteristic structural component of lipid rafts. Cholesterol depletion may therefore result in disorganization of these membrane microdomains (Scheiffele et al., 1997). The drug methyl-β-cyclodextrin (MβCD), a cholesterol depleting reagent can reduce the cholesterol content and cause disorganization of lipid rafts efficiently. At the same time, unlike other cholesterol-binding agents that become incorporated into membranes, MβCD is a strictly surface-acting agent and can rapidly remove cholesterol from the plasma membrane.
In this study, we were especially interested in understanding whether cholesterol is required for TGEV infection and if so, whether cholesterol is important as a constituent of the virus, of the host cells or of both. To address this question, we used MβCD as cholesterol depleting agent to treat either TGEV or swine testicle (ST) cells prior to virus infection. Our results show that depletion of cholesterol from either the viral or the cellular membrane resulted in a decrease of the infectivity of TGEV on ST cells. The concentration of MβCD and cholesterol we used in this study do not produce significant adverse effect on cell viability as shown by trypan blue staining (data not shown). The drug concentration and the protocol applied are similar to those described for the analysis of other viruses (Nayak and Hui, 2004; Imhoff et al., 2007). Other members of the family Coronaviridae, MHV, SARS-CoV, HCoV-229E and IBV (Thorp and Gallagher, 2004; Nomura et al., 2004; Li et al., 2007; Imhoff et al., 2007) are sensitive to MβCD treatment of host cells.
Therefore, the importance of cholesterol-rich microdomains appears to be a general feature of the entry mechanism coronaviruses have developed. In the case of TGEV, the cholesterol-dependence is consistent with the presence of porcine aminopeptidase N in detergent-resistant membrane microdomains.
This holds also true for the human coronavirus 229E which interacts with human aminopeptidase N. MHV and SARS-CoV use different receptors, MHVR and ACE2, respectively. Surprisingly, both of these receptors have been reported to be nonraft-proteins (Thorp and Gallagher, 2004; Warner et al.,
2005). However, MHVR has been shown to redistribute to some extent into lipid rafts after interaction with MHV (Choi et al., 2005). Thus, cholesterol-rich microdomains may contribute to coronavirus entry either by providing platforms for efficient virus binding to receptors presented in these membrane domains or by recruiting virus-receptor complexes to promote the entry process. pathway, the content of cholesterol and sphingolipids is lower compared to the plasma membrane. Therefore, the cholesterol content of coronaviruses is expected to be lower than that of viruses budding from the plasma membrane.
Nevertheless, even the membrane of the endoplasmic reticulum has been shown to contain lipid microdomains (Sevlever et al., 1999). At present we do not know how cholesterol in the viral membrane affects virus infectivity.
However, our results raise the possibility that lipid microdomains exist in the membrane of coronaviruses. The low concentration of cholesterol may explain that the infectivity of TGEV is affected by concentrations of MßCD that are lower than those that affect infectivity of viruses like HIV and influenza virus.
Cholesterol treatment of TGEV without prior MβCD treatment did not affect virus infectivity (data not shown). It will be interesting in future studies to confirm the importance of viral cholesterol for other coronaviruses and to analyze the membrane microdomains in the coronavirus envelope.
3.6 Acknowledgements
This work was performed in partial fulfillment of the requirements for the Dr. Vet. Med. degree at University of Veterinary Medicine Hannover, Germany.
X.R. received a scholarship from German Academic Exchange Service (DAAD). Financial supports to X.R. were from National Natural Science Foundation of China (30700590), Heilongjiang Provincial Science and Technology Department (LC06C01) and Harbin Science and Technology Bureau (2006RFLXN004), and Cultivation Fund of the Key Scientific and
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Figure legends and Figures
Fig.1. Depletion of cholesterol from the cellular membrane.
After treatment of ST cells with various concentrations of MβCD, the cellular cholesterol content was determined (Panel A). The recovery of cellular cholesterol was determined after addition of exogenous cholesterol to cells that have been depleted by 12 mM MβCD (Panel B).
Fig.2. Effect of cellular cholesterol depletion and replenishment on TGEV infection
ST and BHK21 cell monolayers were treated with various concentrations of MβCD; subsequently, cells were infected with TGEV or VSV. The 100%
infectivity values of TGEV and VSV represent average plaque numbers of 160 and 100, respectively (A). ST cell monolayers were treated with 12mM MβCD and subsequently exogenous cholesterol was added. TGEV was used to infect the monolayers; the 100% infectivity value corresponds to an average plaque number of 100 (B).
Fig.3. Changes of viral cholesterol content
After the treatment of MβCD at various concentrations, the viral cholesterol content was determined with cholesterol detection kit (Panel A). The MβCD-treated viruses were further subjected to addition of exogenous cholesterol and the virus pellets were used for cholesterol measurement (Panel B).
Fig.4. Effect of viral envelope cholesterol onTGEV infectivity
TGEV and VSV were treated with 0-10mM MβCD for 30 min at various concentrations, and the MβCD-treated viruses were employed to infect the cell monolayers. The 100% infectivity values of TGEV and VSV correspond to average plaque numbers of 180 and 270, respectively (A). TGEV was treated with MβCD at a concentration of 4mM, and replenished with the 0-500 µM exogenous cholesterol for 30 min. The 100% infectivity value corresponds to an average plaque number of 160 (B)
Fig. 1
A: Cholesterol changes of ST cells after MβCD treatment
0 5 10 15 20 25 30 35 40
0 4 6 8 12
Cellular cholesterol content (µM) (µM)content(µM)
MβCD concentration (mM)
Concentration of cholesterol (µM) )
Cellular cholesterol content (µM) (µM)content(µM)
0 5 10 15 20 25 30 35 40 45
0 50 100 200 500
B: Cellular cholesterol changes after addition of exogenous cholesterol
Fig. 2
0 10 20 30 40 50 60 70 80 90 100
0 50 100 200 500
TGEV
B
In fe c ti v it y %
Concentration of cholesterol (µM)
0 20 40 60 80 100 120 140
0 3 6 9 12 15
TGEV VSV
In fe c ti v it y %
A
MβCD concentration (mM)
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
0 0.5 1 2 4
Fig. 3
A: Cholesterol changes of TGEV after MβCD treatment
Virus cholesterol content (µM)
MβCD concentration (mM)
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
0 50 100 200 500
B: Viral cholesterol changes after addition of exogenous cholesterol
Virus cholesterol content (µM)
Concentration of cholesterol (µM)
Fig. 4
CHAPTER 4
General discussion
4 General discussion
4.1 Apical entry of SARS-CoV is consistent with the localization of the