Institut für Virologie
Characterization of the role of the S protein of coronaviruses for virus entry
INAUGURAL – DISSERTATION Zur Erlangung des Grades eines
Doktors der Veterinärmedizin - Doctor medicinae veterinariae -
(Dr.med.vet.)
Vorgelegt von Xiaofeng Ren
Harbin, China
Hannover 2008
1. Gutachter: Univ.-Prof. Georg Herrler 2. Gutachter: Univ.-Prof. Ulrich Neumann
Tag der mündlichen Prüfung: 14. 08. 2008
To my parents
Parts of this work have been published or have been accepted for publication in the following journals:
1. Journal of General Virology, 87(6), 1691-1695, 2006 2. Virus Research, 2008, in press
CONTENT
1 Introduction ··· 1
1.1 An overview on coronaviruses ··· 2
1.1.1 Taxonomy and antigenic groups··· 2
1.1.2 Structural proteins ··· 3
1.1.3 Genome organization and virus replication ··· 7
1.2 Coronavirus cellular receptors ··· 9
1.2.1 Coronavirus receptor usage and viral entry ··· 9
1.2.2 Establishment of polarized epithelial cell models in vitro··· 13
1.2.3 Receptor-associated coronavirus infection of polarized epithelial cells 15 1.3 An overview on lipid rafts ··· 15
1.3.1 Introduction of lipid rafts ··· 15
1.3.2 Composition of lipid rafts··· 16
1.3.3 Cholesterol and raft biogenesis··· 19
1.3.4 Importance of cholesterol for virus infection··· 20
1.4 References··· 21
2 Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for SARS-associated coronavirus··· 39
2.1 Abstract ··· 40
2.2 Results and Discussion··· 40
2.3 Acknowledgements ··· 45
2.4 References··· 45
3 Importance of cholesterol for infection of cells by transmissible gastroenteritis virus··· 52
3.1 Abstract ··· 53
3.2 Introduction ··· 53
3.3 Materials and Methods··· 55
3.4 Results ··· 57
3.5 Discussion··· 58
3.6 Acknowledgements ··· 60
3.7 References··· 61
4 General discussion ··· 69
4.1 Apical entry of SARS-CoV is consistent with the localization of the cellular receptor, ACE2 ··· 70
4.2 Importance of cholesterol for initiating a TGEV infection··· 75
4.3 References··· 78
5 Summary··· 85
6 Zusammenfassung ··· 88
7 Acknowledgements ··· 91
CHAPTER 1
Introduction
1 Introduction
1.1 An overview on coronaviruses 1.1.1 Taxonomy and antigenic groups
The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are large, enveloped, positive-sense single-stranded RNA viruses that replicate in the cytoplasm of animal host cells (Siddell, 1995).
Coronaviruses were recognized as a new virus family in 1968 because their morphology and intracellular budding site distinguished them from other RNA viruses (Tyrrell et al., 1968; Siddell et al., 1983). In general, coronaviruses have been sorted into three groups (termed groups 1, 2, and 3) that differ in host range and pathogenicity (Gorbalenya et al., 2004; Lai and Holmes, 2001;
Perlman and Dandekar, 2005; Weiss and Navas-Martin, 2005; Navas-Martin et al., 2007).
Group 1 includes two human coronaviruses, HCoV-229E and HCoV-NL63, that cause acute respiratory tract infections (Bastien et al., 2005; Bradburne and Somerset, 1972; Wenzel et al., 1974) and several important animal viruses: porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) cause enteric disease, and porcine respiratory coronavirus (PRCV) causes respiratory disease (Kim et al., 2000;
Kim and Chae, 2003); feline coronavirus (FCoV) and feline infectious peritonitis virus (FIPV) causes enteric or systemic disease in cats (Delmas et al., 1994; Kolb et al., 1998; Poon et al., 2005; Vennema et al., 1998;
Navas-Martin and Weiss SR, 2003) and canine coronavirus (CCoV) causes enteric disease in dogs (Pratelli et al., 2004) .
Group 2 consists of human coronavirus, HCoV-OC43, that causes respiratory disease; human enteric coronavirus (HECV) 4408 isolated from a child with acute diarrhea and is antigenically and genetically more closely related to bovine coronavirus (BCoV) (Han et al., 2006); turkey coronavirus (TCoV) causes enteric or respiratory disease in Turkey; porcine haemagglutinating encephalomyelitis virus (HEV) causes enteric, respiratory diseases or central nervous system (CNS) symptoms; bovine coronavirus (BCoV) causes respiratory disease in cows; rabbit coronavirus (RbEVC) causes enteric disease; sialodacryadenitis virus (SDAV) causes CNS
symptoms in rats; murine hepatitis virus (MHV) causes respiratory or enteric disease in mice and may affect several other organs such as liver and CNS (Navas-Martin and Weiss, 2003; Decaro et al., 2007).
Group 3 mainly includes avian coronaviruses, such as avian infectious bronchitis virus (IBV). IBV causes an upper respiratory tract disease in young chickens characterized by tracheal rales, coughing, sneezing, gasping, and nasal discharge (Lee et al., 2002). However, several strains of IBV have been identified as nephropathogenic, and some of these strains cause significant renal lesions and high mortality in affected chickens (Lee et al., 2002).
Severe acute respiratory syndrome (SARS), was first reported in Guangdong Province, China, in late 2002. This disease eventually affected over 32 countries and areas in Asia, America and Europe via a relatively clear transmission routine (Navas-Martin and Weiss SR, 2003; Zhong et al., 2003).
Its typical clinical symptoms consist of high fevers exceeding 38°C or 100.4°F, myalgia, dry nonproductive dyspnea, lymphopenia and infiltrates on chest radiography. The overall mortality rate was about 10%, particularly, the mortality rate in the elderly was as high as 50% (Donnelly et al., 2003; Hon et al., 2003; Peiris et al., 2003a,b; Stadler et al., 2003).
Through the close international collaboration of a network of 13 laboratories in 9 countries, the researchers have confirmed that a novel coronavirus named SARS associated coronavirus (SARS-CoV) is the causative agent of SARS (Drosten et al., 2003). Genomic studies suggest that the genotype of SARS-CoV is not the same as the genotype of the three known types of coronavirus. Serum of patients infected with a Group 1 coronavirus can react with SARS-CoV, but serum from SARS patients does not react with known coronaviruses. Thus, SARS-CoV can be included in Group 4 as a new type of coronavirus (Stadler et al., 2003; Zhong et al., 2003).
The SARS-CoV cannot only infect the respiratory tract, but also the intestinal tissue (Nicholls et al., 2003; Peiris et al., 2003a,b; Leung et al., 2003). In both cases, the epithelial cells are the primary target of SARS-CoV.
1.1.2 Structural proteins
Coronaviruses are enveloped, single-stranded, RNA viruses. The diameter of virions is about 50-200 nm, and usually of 80-120 nm (Oshiro, 1973; McIntosh, 1974; Masters, 2006). The term “corona” refers to the halo
formed by the viral spike (S) protein extending outwards from the envelope.
The S glycoprotein, together with the integral membrane (M) glycoprotein, a minor envelope (E) protein, and the nucleocapsid (N) protein are the important structural proteins of coronavirus (Fig.1).
The S protein (formerly designated E2) of coronaviruses is a very large, N-exo, C-endo (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) transmembrane protein that assembles into trimers (Delmas and Laude, 1990; Song et al., 2004) to form the distinctive viral surface spikes. Morphologically, it is multi-shaped (club-like or pear-shaped) and projects approximate 17–20 nm from the virion surface (Cavanagh, 1995; .McIntosh, 1974), mainly consisting of a thin base that swells to a width of about 10 nm at the distal extremity (Sugiyama and Amano, 1981; Guy et al., 2000; Patel et al., 1982; Masters, 2006).
S protein is inserted into the endoplasmic reticulum (ER) via an amino-terminal signal peptide (Cavanagh et al., 1986). The ectodomain makes up most of the molecule, with only a small carboxy-terminal segment (of 71 or fewer of the total 1162–1452 residues) constituting the transmembrane domain and endodomain. The S molecule is highly glycosylated, and this modification is exclusively N-linked (Holmes et al., 1981; Rottier et al., 1981). The molecular masses of monomers of S protein without glycosylation are 128–160 kDa and increase to a range of 150–200 kDa post glycosylation. So far, about 19-39 potential consensus glycosylation sites are found in the S protein ectodomains (Masters, 2006).
M S
R sM
蛋 核 CA A(nS protein M protein
sM protein Nucleocapsid
RNA N protein
Envelope
Fig. 1. Schematic drawing of a coronavirus particle
A mass spectrometric analysis of the SARS-CoV S protein has shown that at least 12 of the 23 candidate sites are glycosylated in this molecule (Krokhin et al., 2003). For the TGEV S protein, it has been demonstrated that the early steps of glycosylation occur cotranslationally, but that terminal glycosylation is preceded by trimerization, which can be rate-limiting in S protein maturation (Delmas and Laude, 1990). The S protein ectodomain has between 30 and 50 cysteine residues, and within each coronavirus group the positions of cysteines are well conserved (Abraham et al., 1990; Eickmann et al., 2003). It has been reported that glycosylation of TGEV S may assist monomer folding, given that tunicamycin inhibition of high-mannose transfer was found to also block trimerization, currently however, a comprehensive mapping of disulfide linkages has not yet been achieved for coronavirus S protein (Masters, 2006).
Functionally, the S glycoprotein is the major target of neutralizing antibodies (Garwes et al., 1978; Jiménez et al., 1986; Laude et al., 1987), and it is also related to host cell tropism (Jacobs et al., 1986;
Schwegmann-Wessels et al., 2003), interaction with its cellular receptor (Collins et al., 1982; Delmas et al., 1990; Schwegmann-Wessels et al., 2002, 2003), pathogenicity (Krempl et al., 1997; Garwes et al., 1978; Tuboly et al., 1994), fusion (Collins et al., 1982; Spaan et al., 1988; Sune et al., 1990) and hemagglutination activity (Krempl and Herrler, 2001; Krempl et al., 2000; Kwon et al., 1998).
The M (formerly designated E1) protein, a polytopic protein is the most abundant component of coronavirions (Rottier, 1995). When synthesized in the absence of other viral components, M protein tends to accumulate in the Golgi complex as detergent-insoluble, polymeric structures, presumably as part of a retention mechanism (Krijnse-Locker et al., 1992, 1995). The M protein as the major IFN inducing component has been proposed to play a role in innate immune response to coronaviruses (Charley and Laude, 1988; Laude et al., 1992). It is demonstrated that coexpression of M and E proteins allows the formation of pseudoparticles, which exhibit an interferogenic activity similar to that of complete virions (Baudoux et al., 1998). The M proteins of a subset of group 1 coronaviruses (TGEV, FIPV, and CCoV) each contain a cleavable amino-terminal signal sequence (Laude et al., 1987), although this element may not be required for membrane insertion (Kapke et al., 1988; Vennema et
al., 1991). Another anomalous feature of at least one group 1 coronavirus, TGEV, is that roughly one-third of its M protein assumes a topology in which part of the endodomain constitutes a fourth transmembrane segment, thereby positioning the carboxy terminus of the molecule on the exterior of the virion (Risco et al., 1995; Masters, 2006).
The N gene consists of 1149 nucleotides (nt) in length encoding a phosphorylated protein of 382 aa with a molecular weight of 47 (range from 43 to 50 kDa). The N phosphoprotein complexes with the genomic RNA in a beads-on-a-string fashion to form the nucleocapsid (Cavanagh et al., 1994;
Siddell, 1995). The N protein has been believed to be involved in synthesis of coronavirus RNA via interaction with leader RNA sequences located at the amino-terminus or the sequences at the carboxy terminus of genomic RNA (Baric et al., 1988; Nelson et al., 2000; Zhou et al., 1996). After analysis of purified TGEV virions and multiple fractions from infected cells (Calvo et al., 2005), four sites of phosphorylation (Ser9, Ser156, Ser254, and Ser256) were found. Linear epitopes N46, N272 and N321 might display significant antigenicity (Martin-Alonso et al., 1992). TGEV N protein is also associated with the apoptosis of host cells (Eleouet et al., 2000).
The E integral membrane protein (also designated sM) is a small polypeptide, ranging from 8.4 to 12 kDa (76–109 amino acids), that is only a minor constituent of virions. Owing to its small size and limited quantity, E was recognized as a virion component much later than were the other structural proteins, first in IBV (Liu and Inglis, 1991) and then in TGEV (Godet et al., 1992) and MHV (Yu et al., 1994). E protein sequences are extremely divergent among the three coronavirus groups and in some cases, among members of a single group. Nevertheless, the same general architecture can be discerned in all E proteins: a short hydrophilic amino terminus (8–12 residues), followed by a large hydrophobic region (21–29 residues) containing two to four cysteines, and a hydrophilic carboxy-terminal tail (39–76 residues), the latter constituting most of the molecule. It was asserted that the palindromic hairpin configuration of the transmembrane segment is unique to the SARS-CoV E protein, which raises the question of how the other coronavirus E proteins are arranged in the membrane and why the E protein of SARS-CoV should differ (Masters, 2006).
Some coronaviruses may have a second set of projections beneath the
major spikes, of 5–10-nm in length. The shorter projections are now known to be the hemagglutinin-esterase (HE) protein that is found in a subset of group 2 coronaviruses (Masters, 2006).
1.1.3 Genome organization and virus replication
Coronaviral genomes are nonsegmented, single-stranded, positive-sense 28.5-kb RNA molecules. The genomes are among the largest mature RNA molecules known to biology (Masters, 2006). The amino terminus - about two-third of the entire RNA - comprises open reading frames (ORFs) 1a and 1b, encoding the replicase and a number of other proteins. The carboxy terminus - one-third of the genome - comprises the genes encoding the structural and some nonstructural proteins (Enjuanes et al., 2001). The invariant gene order in all members of the coronavirus family is 5'-1a,b-S-E-M-N-3' (Masters, 2006).
However, some engineered rearrangements of the gene order of MHV were found to be completely tolerated by the virus (de Haan et al., 2002)
Coronaviruses initiate an infection via the binding of virions to cellular receptors. This sets off a series of events culminating in the deposition of the nucleocapsid into the cytoplasm, where the viral genome becomes available for translation (Masters, 2006). The positive-sense genome of coronavirus can also serve as the first mRNA after infection and be translated into the enormous replicase polyprotein. Subseqently, the genome is used as a template guided by the replicase to synthesize both progeny genomes and a series of subgenomic mRNAs via negative-strand intermediates. Viral structural and accessory proteins are produced by the translation of the corresponding subgenomic mRNAs (Fig.2). They resemble most eukaryotic mRNAs, in having both 5' caps (Lai and Stohlman, 1981) and 3' poly (A) tails (Lai and Stohlman, 1978; Lomniczi, 1977; Schochetman et al., 1977; Wege et al., 1978).
Structural proteins, S, M, and E, are inserted into the endoplasmic reticulum (ER), from where they transit to the ER-Golgi intermediate compartment, also designated ERGIC. The encapsidation of progeny genomes by N protein forms the nucleocapsids, which can result in virions with the membrane-bound components by budding into the ERGIC. The resulting progeny virions can be exported from infected cells by transport to the plasma membrane in smooth-walled vesicles, or Golgi sacs with undefined mechanisms. A schematic drawing of the coronavirus life cycle is shown in Fig.3. The cell surface S protein can cause the fusion of an infected cell with adjacent, uninfected cells, leading to the formation of large, multinucleate syncytia. Therefore, the spread of infection by coronavirus is independent of the action of extracellular virus, and in this case, the viruses can escape from immune surveillance (Masters, 2006).
Leader sequence Poly(A) UCUAAAC Encoded fragment N
-mRNA3'
ORF1 S ORF3 sM M UTR
+mRNA5' 3'
Genomic mRNA
Subgenomic mRNAs
5'
1
2 S
3 4 sM
5 M 6 N
7 ORF7
Fig. 2. Schematic drawing of the coronavirus genome
1.2 Coronavirus cellular receptors
1.2.1 Coronavirus receptor usage and viral entry
The corresponding receptors of coronaviruses are highly species-specific, and the interaction between coronavirus S protein and viral receptor remains the principal, if not sole, determinant of coronavirus host species range and tissue tropism (Masters, 2006). Nevertheless, there are still some exceptions that not follow this principle. For example, there are several reports concerning the sporadic cross-species transmission of bovine coronavirus, BCoV (Saif, 2004) or the isolation of BCoV related viruses from wild ruminants (Tsunemitsu et al., 1995), domestic dogs (Erles et al., 2003), and a human child (Zhang et al., 1994). Recently, the adaptation of SARS-CoV to the human population has further challenged the principal (Masters, 2006).
In fact, the manipulation of receptor or coronavirus structural proteins (often S protein) is important for understanding the receptor-related actions between coronaviruses and hosts. By expressing an identified receptor in nonpermssive cells may make the cells susceptible to the corresponding
Fig. 3. Schematic drawing of the life cycle of coronaviruses, adopted from Masters, 2006.
coronaviruses, leading to the finding of viral receptor or cellular determinant of virus infection (Delmas et al., 1992; Dveksler et al., 1991; Li et al., 2003, 2004;
Mossel et al., 2005; Tresnan et al., 1996; Yeager et al., 1992). The engineered swapping of S protein ectodomains has been shown to change the in vitro host cell species specificity between MHV and FIPV (Kuo et al., 2000; Haijema et al., 2003). Exchange of the relevant regions of S protein ectodomains has been shown to transform a strictly respiratory isolate of TGEV into a more virulent, enterotropic strain (Sanchez et al., 1999). Replacement of the S protein ectodomain of MHV strain A59 by that of other MHV strains caused the virus to acquire the highly virulent neurotropism of MHV strain 4 (Phillips et al., 1999) or the highly virulent hepatotropism of MHV strain 2 (Navas et al., 2001).
Most of group 1 coronaviruses use the species-specific aminopeptidase N (APN) as a receptor (Delmas et al., 1992; Tresnan et al., 1996; Yeager et al., 1992). APN (also designated CD13) is a cell-surface, zinc-binding protease that contributes to the digestion of small peptides in respiratory and enteric epithelia; and it is also found in human neural tissue that is susceptible to HCoV-229E (Lachance et al., 1998). The APN molecule is a homodimer; each monomer has a C-exo, N-endo membrane orientation and is heavily glycosylated (Masters, 2006; Tusell et al., 2007). There are some overlaps between the catalytic domain of hAPN and the binding site for HCoV-229E (Yeager et al., 1992). Although APN as receptors for many coronaviruses are species-specific (Delmas et al., 1994a; Kolb et al., 1996), the fAPN can also serve as a receptor for CCoV, TGEV, and HCoV-229E in addition to FIPV (Tresnan et al., 1996). These contrasting properties have been used as the framework for dissecting the basis of species-specific or -nonspecific function, through the construction and analysis of chimeric receptors (Benbacer et al., 1997; Delmas et al., 1994a; Hegyi and Kolb, 1998; Kolb et al., 1996, 1997;
Masters, 2006). Not all group 1 coronaviruses use APN as a receptor, however, it has been proposed that one subset of FIPV strains uses a different receptor, since an antibody to fAPN blocked replication of type II strains of FIPV but not replication of type I strains of FIPV (Hohdatsu et al., 1998). This conclusion is consistent with the observation that there is greater sequence divergence between type I FIPV S proteins and type II FIPV S proteins than there is between type II FIPV S proteins and the S proteins of CCoV or TGEV
(Herrewegh et al., 1998; Motokawa et al., 1996).
In the case of TGEV and PEDV, two porcine coronaviruses grow in mutually exclusive sets of cell lines derived from different species (Hofmann and Wyler, 1988). It was believed that the major receptor for PEDV may differ from that for TGEV. Recently, however, it has been shown that pAPN facilitates cellular entry of PEDV (Oh et al., 2003) and expression of pAPN on PEDV nonpermissive cells resulted in the infection by PEDV (Li et al., 2007), supporting the notion that pAPN also serves as a receptor for PEDV. However, more work is needed to clarify the binding process between PEDV S protein and its functional receptor and especially the question why PEDV cannot be propagated in ST cell, a porcine APN-expressing and TGEV-susceptible cell line.
Another exception to the utility of APN as a receptor for group 1 coronaviruses is a new human coronavirus, HCoV-NL63 that uses the same receptor as SARS-CoV, namely ACE2 rather than hAPN to initiate infection (Hofmann et al., 2005). HCoV-NL63 causes a much milder respiratory disease than does SARS-CoV.
The MHV receptor (formerly MHVR1, now designated mCEACAM1) is a member of the carcinoembryonic antigen (CEA) family, a group of proteins within the immunoglobulin (Ig) superfamily. CEACAM1 was the first receptor discovered for a coronavirus and is one of the first receptors found for any virus (Williams et al., 1990, 1991). The wide range of pathogenicity of MHV in mice is thought to result from the interactions of S proteins of different virus strains with the tissue-specific spectra of receptor variants displayed in mice having different genetic backgrounds. The CEACAM1 may be the only biologically relevant receptor for MHV (Smith et al., 1991; Hemmila et al., 2004) and CEACAM2 cannot be used as an alternative receptor in vivo, although the latter is the product of the other murine Ceacam gene family member (Nedellec et al., 1994).
Other group 2 coronaviruses use different receptors. The rat coronaviruses RCoV and SDAV, although closely related to MHV and able to grow in some of the same cell lines as does MHV, do not gain entry to cells via mCEACAM1 (Masters, 2006). BCoV is phylogenetically close to MHV, but the two viruses do not show any relatedness in usage of hosts or susceptible cells.
The exclusively identified cell attachment factor for BCoV is 9-O-acetylated sialic acid (Schultze et al., 1991).
SARS-CoV is phylogenetically most distant from all other group 2 coronaviruses, and it uses angiotensin-converting enzyme 2 (ACE2) as a cellular receptor that is unrelated to CEACAMs. This receptor was identified through the use of a SARS-CoV S1-IgG fusion protein to immunoprecipitate membrane proteins from Vero E6 cells, an African green monkey kidney cell line that is the best in vitro host for SARS-CoV (Li et al., 2003). Supporting evidence included that binding of S1-IgG to Vero E6 cells was inhibited by soluble ACE2 protein but not by a related protein, ACE1. Expression of cloned cDNA for ACE2 rendered nonpermissive cells susceptible to infection by SARS-CoV (Li et al., 2003). ACE2 identified by the expression cloning technique was shown to render cells infectable by a retroviral pseudotype carrying the SARS-CoV S protein (Wang et al., 2004).
ACE2 is a zinc-binding carboxypeptidase that is involved in regulation of heart function. It is an N-exo, C-endo transmembrane glycoprotein with a broad tissue distribution (Masters, 2006). Active-site mutants of ACE2 showed no detectable defects in binding to SARS-CoV S protein (Moore et al., 2004) or in promoting S protein-mediated syncytia formation (Li et al., 2003), suggesting that ACE2 catalytic activity is not required for receptor function. This conclusion needs to be verified by direct SARS-CoV infection, however.
Recently solved x-ray structures for ACE2 have revealed that a large conformational change is induced by the binding of an inhibitor in the active site of the enzyme (Towler et al., 2004). Although this finding raised the possibility of interfering with the initiation of infection, the inhibitor does not affect S protein binding or receptor function of ACE2 (Li et al., 2005).
Various cell lines have been classified with respect to their permissivity or nonpermissivity to SARS-CoV (Gillim-Ross et al., 2004; Giroglou et al., 2004;
Mossel et al., 2005). It has been shown that human ACE2 is a much better receptor than murine ACE2 as far as the binding of the S protein of SARS-CoV is concerned (Li et al., 2004) and nonpermissive cells were shown to become permissive after expression of human ACE2 (Mossel et al., 2005). In addition, human CD209L (also designated L-SIGN or DC-SIGNR), a lectin family member, has also been found to act as a receptor for SARS-CoV, but it has
much lower efficiency compared with ACE2 (Jeffers et al., 2004). A related lectin, DC-SIGN, was identified as a coreceptor, since it was able to transfer the virus from dendritic cells to susceptible cells; however, DC-SIGN could not act as receptor on its own (Marzi et al., 2004; Yang et al., 2004).
IBV, the avian coronavirus of group 3, causes severe respiratory disease in chickens, and its receptor has not been identified. Feline kidney cells that express fAPN were permissive to IBV and the hamster cells became permissive to IBV after transfection with a fAPN cDNA suggesting that the feline APN molecule might be a functional receptor for IBV (Miguel et al., 2002).
Recently however, other experiments showed that both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells; however, fAPN expression does not rescue infection by the prototype IBV strain Mass41. APN-expressing BHK-21 cells are slightly susceptible to certain IBV strains, but this level of infection is not increased by fAPN expression (Chu et al., 2007). Therefore, the receptor usage of IBV may be strain-dependent.
It is also documented that heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus Beaudette (Madu et al., 2007) and sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus (Winter et al., 2006, 2008).
1.2.2 Establishment of polarized epithelial cell models in vitro
The primary targets of both SARS-CoV and TGEV are epithelial cells, and they are usually organized in a polarized way. The plasma membrane of polarized epithelial cell can be distinguished into two distinct domains. One is designated apical domain that is directed towards the external environment and the other is the basolateral domain that faces the internal tissues and blood vessels. Both domains differ in composition and function, which is achieved by at least two mechanisms. One is the tight junction located in the border area between the apical and basolateral domains. The other is vectorial transport (Compans and Herrler, 2005). Therefore, some proteins are localized in different domains of the epithelial cells. Utilizing a special culture apparatus, the apical and basolateral domains can be manipulated separately by seeding the differentiated cells on special membrane filters (Fig.4).
To perform a localization or functional analysis for proteins including viral receptors, some labeling reagents or viruses can be added from either of the two domains. Freshly cultured epithelial cells might not be necessary to be confluent and thus might not be polarized totally. The polarity and integrity of the cell monolayer therefore need to be confirmed by detecting the transepithelial resistance (TER) value (using a special apparatus). Disrupting the tight junction using chemical reagents such as EGTA or adding different colored medium in either of the cultural chambers allows to monitor the establishment or loss of transepithelial resistance. It should be noted that only the well-differentiated and polarized epithelial cells can be used as convincing in vitro models for initiating related studies including cellular receptor localization and virus infection analysis.
Fig. 4. Schematic drawing of differentiation of polarized epithelial cells in vitro
1.2.3 Receptor-associated coronavirus infection of polarized epithelial cells
The apical and basolateral domains of epithelial cells differ in lipid and protein compositions (Rossen et al., 1994). The polarity of these cells affects both the early and late stage of infection, i.e. viruses may enter into and exit from a cell either via the apical or the basolateral membrane.
Evidently, the surface expression of a cellular receptor is a critical determinant of the virus infection with respect to the attachment and penetration process. The entry site of TGEV has been analyzed by infecting filter-grown LLC-PK1 cells from the apical and basolateral sides, respectively.
The results show that the virus entered the cells from both sides at the early stage after plating. With the development of cell polarity, however, the entry was found to be restricted to the apical membrane and could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N (Rossen et al., 1994). Analysis of radioactively labeled viral proteins and authentic virus particles, together with the electron microscopy analysis of newly synthesized virions, supported the preferential release of TGEV from the apical plasma membrane (Rossen et al., 1994, 1995).
1.3 An overview on lipid rafts 1.3.1 Introduction of lipid rafts
Eukaryotic cells are surrounded by a membrane designated the plasma membrane. Lipid rafts, also designated detergent-resistant domains (DRMs), are plasma membrane microdomains characterized by insolubility in cold nonionic detergents such as Triton X-100 or Brij-98 and by enrichment of cholesterol and sphingomyelin (Glebov and Nichols, 2004; Kenworthy and Edidin, 1998). Lipid rafts are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms.
The lipid rafts range from a few nanometers to a few hundred nanometers in diameter, and most researchers agree that, if rafts exist, they are extremely small (25–100 nm) and transient, and represent 50% of cellular membrane, approximately (Glebov and Nichols, 2004; McConnell, 2005). Certain proteins reside preferentially in rafts, whereas others are recruited or excluded from the rafts upon cell activation (Glebov and Nichols, 2004; Kenworthy and Edidin,
1998; McConnell, 2005; Veatch and Keller, 2005; Metropolis et al., 1953;
Stanley, 1971). The lipid rafts have heterogeneous protein compositions: some are enriched in caveolin and others concentrate clathrin and GPI-anchored proteins or dynamin (Glebov and Nichols, 2004; Kenworthy and Edidin, 1998;
Yethiraj and Weisshaar, 2007).
Caveolae (“little caves”) are a specialized type of raft characterized by 50-100 nm invaginations in the plasma membrane (Deurs et al., 2003). The distinctive pitted cytomorphology exhibited by caveolar rafts arises from the structural contribution of membrane proteins of the caveolin family (Drab et al., 2001; Park et al., 2002; Liu et al., 2002). Caveolins regulate signal transduction processes by direct protein-protein interaction and by facilitating molecular sequestration within caveolae (Liu et al., 2002). Lipid rafts that do not contain caveolins, and which consequently do not form invaginated membrane structures, are also isolated from cells using identical approaches to those used for caveolar rafts (Zhuang et al., 2005).
Lipid rafts are believed to exert various functions by providing a specialized microenvironment in which the relevant molecules for the initiation of the specific biological processes are partitioned and concentrated (Brown and London, 2000). The rafts play important roles in signal transduction (Cheng et al., 2001; Janes et al., 2000), form receptor complexes, act as platforms for cell–cell interactions (Viola et al., 1999; Moran and Miceli, 1998), and have also been implicated in membrane trafficking (Brown and London, 1998; Veatch and Keller, 2005; Verkade and Simons, 1997).
1.3.2 Composition of lipid rafts
Lipid rafts are mainly composed of sphingolipids and cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. These assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase (Brown and London, 1998;
Simons and Ehehalt, 2002).
As above-mentioned, the saturated acyl chains of the sphingolipids in rafts promote their dense packing in the membrane while cholesterol fills up the
space between adjacent sphingolipid head groups and fatty acid chains (Schroeder et al., 1994). This tight packing structure gives rise to the characteristic detergent resistance of lipid rafts, and these DRMs literally floated like rafts to the top of the preparations after sucrose gradient centrifugation (Schuck et al., 2003; Lingwood and Simons, 2007; Schroeder et al., 1994). This makes it possible to separate the lipid rafts and associated proteins from other non-raft cellular components using simple physiochemical methods.
Currently, membrane proteins are assigned to three categories: those that are mainly found in the rafts, those that are present in the liquid-disordered phase, and those that represent an intermediate state, moving in and out of rafts. Constitutive raft residents include glycophosphatidylinositol-anchored (GPI-anchored) proteins; doubly acylated proteins, such as tyrosine kinases of the Src family, Gsubunits of heterotrimeric G proteins, and endothelial nitric oxide synthase (eNOS); cholesterol-linked and palmitate-anchored proteins like Hedgehog (Simons and Ehehalt, 2002; Jeong and McMahon, 2002). A proposed lipid raft model is shown in Fig.5.
Fig. 5. Schematic drawing of the lipid raft adopted from the center of
media archive of Wikimedia Commons
(http://de.wikipedia.org/wiki/Lipid_Raft) with modifications.
Note: A: Intracellular space or cytosol; B: Extracellular space or vesicle/Golgi apparatus lumen; Black framed region: Lipid raft;
1: GPI-anchored protein; 2: Glycosylation modifications (on glycoproteins and glycolipids); 3: Cholesterol; 4: Lipid raft associated transmembrane protein.
1.3.3. Cholesterol and raft biogenesis
Cholesterol is an amphipathic molecule, meaning that, like phospholipids, it contains a hydrophilic and a hydrophobic portion. Cholesterol's hydroxyl (OH) group aligns with the phosphate heads of the phospholipids (Alberts et al., 2002). The remaining portion of it tucks into the fatty acid portion of the membrane. Cholesterol is therefore thought to serve as a spacer between the hydrocarbon chains of the sphingolipids and to function as a dynamic glue that keeps the raft assembly together (Simons and Toomre, 2000).
Cholesterol partitions between the raft and the nonraft phase, having higher affinity to raft sphingolipids than to unsaturated phospholipids. Removal of raft cholesterol leads to dissociation of most proteins from rafts and may render them nonfunctional. If cholesterol is extracted by methyl-beta-cyclodextrin or complexed by saponin, the raft proteins usually, but not always, become detergent-soluble. Lipid rafts are believed to be first assembled in the Golgi complex in mammalian cells (Brown and London, 1998).
Cholesterol is synthesized in the endoplasmic reticulum (ER), as is ceramide, the hydrophobic backbone of sphingolipids. However, most of the sphingolipid head groups are attached to ceramide in the Golgi complex, where raft assembly takes place (van Meer, 1989). There is an increasing concentration of cholesterol and sphingolipids from the ER to the plasma membrane. This increase seems to be achieved by excluding lipid rafts from the retrograde traffic between the Golgi complex and the ER (Brugger et al., 2000). Thus, lipid rafts are moved forward from the Golgi complex to the plasma membrane, where they concentrate but also spread into the endocytic recycling pathways (Mukherjee and Maxfield, 2000). Cholesterol and sphingolipid concentrations are tightly regulated and limit the supply of lipid rafts to organelles supplied by the Golgi apparatus (Simons and Ehehalt, 2002).
Cholesterol can make up nearly half of the cell membrane, however, it is smaller and weighs less than other molecules in the cell membrane, it makes up a lesser proportion of the cell membrane's mass, usually roughly 20 percent.
Cholesterol is also present in membranes of organelles inside the cells, although it usually makes up a smaller proportion of the membrane. For
example, the mitochondrion, the so-called "power-house" of the cell, contains only three percent cholesterol by mass, and the endoplasmic reticulum, which is involved in making and modifying proteins, is six percent cholesterol by mass (Alberts et al., 2002).
In the plasma membrane, cholesterol is closely attracted to part of the fatty acid chain on the nearest phospholipid. This helps to slightly immobilize the outer surface of the membrane and to make it less soluble to very small water-soluble molecules that could otherwise pass through more easily.
Without cholesterol, cell membranes would be too fluid, not rigid enough, and too permeable to some molecules. While cholesterol adds rigidness and integrity to the plasma membrane and prevents it from becoming overly fluid, it also helps to maintain its fluidity. High concentrations of cholesterol help to separate the phospholipids so that the fatty acid chains cannot come together and cyrstallize. Therefore, cholesterol helps to prevent extremes -- whether too fluid, or too rigid -- in the consistency of the cell membrane (Alberts et al., 2002).
1.3.4 Importance of cholesterol for virus infection
Accumulating evidence suggest the involvement of membrane lipid rafts in virus life cycles (Simons and Ehehalt, 2002). The importance of cholesterol in the entry of nonenveloped viruses has been demonstrated for simian virus 40 (SV40), rotavirus, enterovirus and rhinovirus (Suzuki and Suzuki, 2006). Entry of enveloped viruses into cells involves binding to specific receptors and fusion of the viral membrane with a cellular membrane.
Successful virus entry may require cholesterol in either of the two membranes involved or in both. Previous data show that the infectivity of influenza virus is sensitive to cholesterol depletion from the viral membrane (Smith and Helenius, 2004). More recently it has been shown that canine distemper virus infection also requires cholesterol in the viral envelope (Imhoff et al., 2007). In contrast, murine leukemia virus, Ebola virus and Marburg virus are sensitive to cholesterol depletion from the cellular membrane (Bavari et al., 2002; Lu et al., 2002). In the case of human immunodeficiency virus (HIV) and herpes simplex virus, cholesterol is required in both membranes (Smith and Helenius, 2004; Nayak and Hui, 2004), while vesicular stomatitis virus (VSV) replication is not affected by cholesterol depletion.
Current data indicate that depletion of cellular cholesterol by the drug methyl-β-cyclodextrin (MβCD), a cholesterol depletion reagent inhibits virus entry of the coronaviruses mouse hepatitis virus (Thorp and Gallagher, 2004;
Choi et al., 2005), severe acute respiratory syndrome (SARS)-coronavirus (Li et al., 2007), human coronavirus 229E (Nomura et al., 2004) and avian infectious bronchitis virus (Imhoff et al., 2007).
As coronaviruses differ widely in their hosts and tissue tropism and use quite different cellular receptors for virus entry, we investigated, for the first time, the requirement of cholesterol for a porcine coronavirus, transmissible gastroenteritis virus.
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