Cellular Entry of the SARS Coronavirus: Implications for Transmission, Pathogenicity and Antiviral Strategies

  • Ilona Glowacka
  • Stephanie Bertram
  • Stefan PöhlmannEmail author


A novel coronavirus was identified as the causative agent of the lung disease severe acute respiratory syndrome (SARS). The outbreak of SARS in 2002/2003 was associated with high morbidity and mortality and sparked international research efforts to develop antiviral strategies. Many of these efforts focussed on the viral surface protein spike (S), which facilitates the first indispensable step in the viral replication cycle, infectious entry into target cells. For infectious cellular entry to occur, the S protein must engage a cellular receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). The interface between ACE2 and S protein, which has been characterized at the structural level, constitutes a key target for vaccines and inhibitors, and is believed to be an important determinant of viral pathogenesis and interspecies transmission. In this chapter, we will discuss how SARS-S mediates cellular entry and we will review the implications of this process for SARS coronavirus (SARS-CoV) transmission, disease development and antiviral intervention.


Acute Respiratory Distress Syndrome Severe Acute Respiratory Syndrome Severe Acute Respiratory Syndrome Receptor Binding Domain Target Cell Membrane 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1.1 Introduction

The emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV) in Guangdong Province, China, in 2002, and its subsequent spread in Asia and Canada clearly exemplified the vulnerability of societies and economies to a novel, highly pathogenic respiratory agent (Stadler et al. 2003; Peiris et al. 2003b). The outbreak, which was halted solely by the quarantine of exposed individuals and the use of conventional prevention measures such as surgical masks, was paralleled by an international, collaborative scientific effort to develop means for therapeutic and preventive intervention (Peiris et al. 2004; Stadler and Rappuoli 2005). The basis for the development of successful antiviral strategies is a thorough understanding of the molecular biology underlying viral amplification and pathogenesis, and many significant discoveries have been made in the SARS field since the identification of the virus early in 2003 (Drosten et al. 2003; Ksiazek et al. 2003; Peiris et al. 2003a). Several of these findings provided important insights into the structure and function of the viral spike (S) protein, which is used by the virus as the key to bind and enter host cells (Hofmann and Pöhlmann 2004). The most well-known examples are the identification of angiotensin-converting enzyme 2 (ACE2) as the host factor which is engaged by the viral S protein for infectious entry into cells, and the elucidation of the structure of the S protein receptor binding domain (RBD) in complex with ACE2 (Li et al. 2003, 2005a). These findings have major implications not only for vaccine and inhibitor development but also for our understanding of the SARS zoonosis, since adaptation of SARS-S to robust usage of human ACE2 was probably of key importance for efficient SARS-CoV spread in humans (Li et al. 2005a, 2005c). In this chapter, we will discuss how SARS-CoV gains access to target cells and how this process can be inhibited. In addition, we will review how the molecular interactions underlying SARS-CoV entry impact viral pathogenesis and interspecies transmission.

1.2 The Spike Protein: Key to the Host Cell

The SARS-Sprotein is a type I transmembrane protein, which comprises 1,255 amino acids and contains 23 consensus signals for N-linked glycosylation (Hofmann and Pöhlmann 2004). S protein is synthesized in the secretory pathway of infected cells. It contains an N-terminal signal sequence, which mediates import of the nascent protein into the endoplasmatic reticulum, where the protein is folded and modified with mannose-rich carbohydrates. Upon transport of the protein into the Golgi apparatus, most, if not all, of the high-mannose carbohydrates are processed into complex glycans (Nal et al. 2005). Evidence of O-glycosylation of SARS-S has not been reported. A novel dibasic ER retrieval motif in the cytoplasmic tail of SARS-S promotes accumulation of the S protein at the ER–Golgi intermediate compartment and the Golgi region (McBride et al. 2007), the sites where progeny particles are assembled (Stertz et al. 2007; Siu et al. 2008). Formation and budding of new particles are driven by the membrane protein (M), the envelope protein (E) and the nucleocapsid protein (N) (Huang et al. 2004; Hsieh et al. 2005; Siu et al. 2008); interactions with the M protein might facilitate S protein incorporation into particles. Trimers of the S protein protrude from the viral envelope and provide virions with a crown (Lat. corona) -like appearance, from which the name “coronaviruses” is derived.

The domain organization of SARS-S resembles that of several well-characterized viral membrane proteins, such as influenza virus hemagglutinin (HA) and human immunodeficiency virus (HIV) envelope protein (Env) (Hofmann and Pöhlmann 2004). These proteins employ comparable strategies to facilitate fusion of viral and host cell membranes and are termed class I fusion proteins (Kielian and Rey 2006). They are distinguished from class II fusion proteins (Kielian 2006), found, for example, on flavi- and alphaviruses, by their distinct spatial organization and the particular configuration of the functional elements required for fusion with target cells: class I fusion proteins are inserted perpendicular to the viral membrane and contain an N-terminal surface unit (SU) and a C-terminal transmembrane unit (TM). The globular SU interacts with cellular receptors, while the TM promotes fusion of the viral and host cell membrane (Kielian and Rey 2006). The latter process depends on the presence of a fusion peptide and two helical regions (HR), conserved elements which are intimately involved in the membrane fusion process (Fig. 1.1), as discussed below. The S protein and the aforementioned fusion proteins are adapted to usage by different cellular receptors. Therefore, the SU (termed S1) of SARS-S does not exhibit appreciable sequence homology to the respective sequences of other class I fusion proteins. In contrast, the functional elements in TM, particularly the HRs, are conserved between different class I fusion proteins. Consequently, the TM (termed S2) of SARS-S shares homology with the corresponding sequences of other viral fusion proteins (Hofmann and Pöhlmann 2004), which has important implications for development of antiviral strategies, as discussed below.
Fig. 1.1

Domain organization of coronavirus S proteins (adapted from Hofmann and Pöhlmann 2004). The position of the S protein open reading frame in the SARS-CoV genome is indicated in the upper panel. Coronavirus S proteins exhibit a domain organization characteristic for class I fusion proteins. The domain organization of prototype class I fusion proteins, the HIV envelope protein, and the influenza virus HA is shown below. A signal peptide is located at the N terminus and mediates import of the nascent protein into the secretory pathway of infected cells. The surface unit S1 contains a receptor binding domain (RBD), which allows engagement of cellular receptors for infectious entry. The transmembrane unit (S2) harbors functional elements pivotal to membrane fusion: a fusion peptide, two helical regions, and a transmembrane domain. Proteolytic cleavage into the S1 and S2 subunits by host-cell proteases is indicated by a triangular arrow. AIBV: avian infectious bronchitis virus; hCoV: human CoV; HR: helical region; MHV: murine hepatitis virus; SARS: severe acute respiratory syndrome

1.3 The Attachment Factors DC-SIGN and DC-SIGNR: Enhancers or Inhibitors of SARS-CoV Infection?

The interaction of SARS-S with ACE2 is the first indispensable step in the entry cascade, as discussed below. It needs to be noted, however, that SARS-S also binds to other cell surface factors and these interactions, although being ultimately dispensable for infectious entry, might profoundly alter infection efficiency. Thus, it is well established that the calcium-dependent (C-type) lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209; Geijtenbeek et al. 2000) and the related protein DC-SIGNR (L-SIGN, CD209L; Pöhlmann et al. 2001; Bashirova et al. 2001) bind to SARS-S (Marzi et al. 2004; Yang et al. 2004; Jeffers et al. 2004; Khoo et al. 2008) in a glycan-dependent fashion (Shih et al. 2006; Han et al. 2007). DC-SIGNR was detected in the lung on ACE2-positive, SARS-CoV-infected cells and on uninfected bystander cells (Chan et al. 2006), while DC-SIGN expression was found to be induced upon SARS-CoV infection (Yen et al. 2006), suggesting that these lectins might have ample opportunity to capture SARS-CoV in infected individuals.

Despite the potentially important role of DC-SIGN/R in SARS-CoV infection, the consequences of DC-SIGN/DC-SIGNR (collectively referred to as DC-SIGN/R) engagement by SARS-S for viral infectivity are largely unclear. One group suggested that DC-SIGN/R function as bona fide receptors which facilitate viral entry into otherwise nonpermissive HeLa cells (Han et al. 2007). DC-SIGNR was also identified in a functional screen for receptors used by SARS-CoV for cellular entry (Jeffers et al. 2004), further suggesting that DC-SIGNR might support viral entry, at least under certain conditions and probably with low efficiency. In contrast, three other studies failed to detect an appreciable receptor function for DC-SIGN/R but suggested that these proteins might enhance infectious entry into ACE2-expressing cell lines (Marzi et al. 2004; Yang et al. 2004; Shih et al. 2006). Finally, and in contrast to all aforementioned studies, Chan and co-workers provided evidence that DC-SIGNR plays a protective role in SARS-CoV infection (Chan et al. 2006). Thus, it was demonstrated that DC-SIGNR-dependent uptake of SARS-CoV into cell lines might lead to viral degradation and might thus reduce viral infectivity for target cells (Chan et al. 2006). In agreement with this finding, evidence was obtained that the combination of certain DC-SIGNR allelic variants, which resulted in reduced SARS-CoV uptake in cell culture, was associated with increased risk of SARS-CoV infection in humans (Chan et al. 2006), albeit these findings are not undisputed (Tang et al. 2007; Zhi et al. 2007). In any case, most functional studies described above have in common that they were carried out with cell lines, which do not adequately model type II pneumocytes, the major targets of SARS-CoV infection (Hamming et al. 2004; Ding et al. 2004; To and Lo 2004; Mossel et al. 2008), and further work with primary lung epithelium is required to help to elucidate the role of DC-SIGN/R in SARS-CoV infection. Notably, a single study examined the impact of DC-SIGN/R-specific antibodies on viral spread in primary human airway epithelium cultured at the air–liquid interface and observed no inhibition (Sims et al. 2008), although it was not investigated if these lectins were indeed expressed by the cells examined. Finally, it is worth mentioning that SARS-S binds to lectins other than DC-SIGN/R, such as the C-type lectin LSECtin (Gramberg et al. 2005) which is largely co-expressed with DC-SIGNR, and the consequences of these interactions for viral amplification have not been determined. Collectively, it is clear that binding to DC-SIGN/R and related lectins has the potential to modulate viral spread in vivo. It remains to be determined, however, if lectin binding augments or suppresses viral replication. Recently described knock-in mice for human DC-SIGN (Schaefer et al. 2008) or SIGNR1 (a murine homologue of human DC-SIGN) knock-out mice (Lanoue et al. 2004) might be useful to clarify these questions.

1.4 The Two Faces of ACE2: SARS-CoV Receptor and Protector Against Lung Damage

In contrast to attachment factors, cellular receptors are indispensable for infectious viral entry. In order to discover such factors, several laboratories used the soluble SARS-S1 subunit for co-immunoprecipitation of cellular binding partners. A milestone study by Li and colleagues identified the carboxypeptidase ACE2, an integral part of the renin–angiotensin system (see below), as a high-affinity SARS-S interactor (Li et al. 2003). Ectopic expression of ACE2 on barely permissive 293T cells facilitated efficient SARS-S-dependent cell–cell and virus–cell fusion (Li et al. 2003), suggesting that ACE2 might play an important role in SARS-CoV entry. Similar results were obtained by an independent study (Wang et al. 2004), which used a comparable approach to identify cellular binding partners of SARS-S. Subsequently, it was shown that endogenous expression of ACE2 correlates with susceptibility to SARS-CoV infection of cell lines (Nie et al. 2004; Hofmann et al. 2004a) and that ectopic expression of ACE2 facilitates SARS-S-driven infection of otherwise nonsusceptible cells (Mossel et al. 2005). Moreover, it was demonstrated that SARS-CoV infects ACE2-positive type II pneumocytes and ACE2-positive cells in the intestinal epithelium (Hamming et al. 2004; Ding et al. 2004; To and Lo 2004; Chan et al. 2006; Mossel et al. 2008), albeit ACE2-independent infection of target cells has also been suggested (Gu et al. 2005; Gu and Korteweg 2007). Finally, knock-out of ACE2 in mice was found to largely abrogate susceptibility to SARS-CoV infection (Kuba et al. 2005), indicating that ACE2 functions as a bona fide SARS-CoV receptor, which is necessary and sufficient for infectious entry into target cells.

1.4.1 The Structure of the Interface Between SARS-S and ACE2

A thorough understanding of the interface between SARS-S and ACE2 is key to the development of antiviral strategies targeting viral entry. The domains and amino acid residues in SARS-S and ACE2, which contribute to the efficient interaction of these proteins, were initially mapped by mutagenic analyses. These studies identified amino acids 318–510 in SARS-S as an independently folded RBD, which binds to ACE2 with higher affinity than the full length S protein (Xiao et al. 2003; Wong et al. 2004; Babcock et al. 2004). The RBD was also shown to be the major target of neutralizing antibodies (He et al. 2004a, 2004b, 2005), and several residues within amino acids 450–490 were suggested to be critical for optimal ACE2 engagement (Wong et al. 2004; Li et al. 2005c). In addition, exploitation of species-specific differences in murine, rat and human ACE2 allowed the mapping of certain amino acid residues, particularly L353, as important for receptor function (Li et al. 2004, 2005c). These results were supported and extended by the subsequent solution of the structure of the RBD in complex with ACE2 (Li et al. 2005a): the RBD consists of a core (a five-stranded antiparallel β-sheet), and an extended loop, which contains all amino acids making contacts with ACE2. The extended loop, also termed receptor-binding motif (RBM), comprises amino acids 424–494 (Li et al. 2005a), and thus includes the residues defined by mutagenic analysis to be important for SARS-S interactions with ACE2 (Wong et al. 2004; Li et al. 2005c). The RBM contacts the N-terminal helix of ACE2 and the loop between helices α2 and α3. Moreover, a portion of the RBM inserts between a short helix in ACE2 (amino acids 329–333) and a β-hairpin at ACE2 residue L353, supporting the previously postulated contribution of L353 to appropriate spike–receptor interactions (Li et al. 2005c). Conformational changes inherent to the peptidase activity of ACE2 do not impact the availability of the S protein binding site (Li et al. 2005a), in agreement with the observation that an ACE2 inhibitor which blocks peptidase activity and arrests ACE2 in a closed conformation does not inhibit SARS-S-dependent entry (Towler et al. 2004; Li et al. 2005c). Collectively, the functional and structural studies defined amino acids in SARS-S and ACE2, which facilitate the tight association of these proteins. In addition, the results highlighted that natural variation of these sequences might have important implications for SARS-CoV transmission and pathogenicity, as discussed below.

1.4.2 Sequence Variations at the SARS-S/ACE2 Interface Might Impact Viral Transmission and Pathogenicity

Horseshoe bats harbor SARS-CoV-related viruses and might constitute the natural reservoir of SARS-CoV (Lau et al. 2005; Li et al. 2005b). However, the sequence homology between bat and human viruses is limited (Lau et al. 2005; Li et al. 2005b). Thus, the S protein of animal viruses does not contain an RBM-like sequence and does not use ACE2 for cellular entry (Ren et al. 2008). It is therefore probable that SARS-CoV was introduced into the human population via an intermediate host, and palm civets, which harbor viruses with high sequence homology to human SARS-CoV, are possible candidates (Guan et al. 2003; Song et al. 2005). Notably, the S proteins of human viruses from the 2002/2003 epidemic bind human ACE2 with much higher efficiency than their palm civet counterparts (Li et al. 2005c), indicating that efficient spread in humans required adaptation of the SARS-S sequence. Indeed, sequence comparison revealed that the civet RBD contains four amino acid changes relative to the human sequence. Two of these changes are located outside the RBM and do not impact receptor interactions (Li et al. 2005c). In contrast, the remaining two changes, N (human) to K (palm civet) at position 479 and T (human) to S (palm civet) at position 487, afflicted residues making direct contact with ACE2 and significantly decreased binding to human ACE2 (Li et al. 2005a, 2005c). Thus, N479 and T487 might be required for efficient spread in and between humans (Li et al. 2005a, 2005c; Li 2008). Interestingly, viruses isolated from sporadic SARS cases in the winter of 2003/2004, which were not associated with severe disease or human-to-human transmission, contained a serine at position 487 (Li et al. 2005a), further indicating that this amino acid might play a key role in human-to-human transmission and viral pathogenicity.

The potential for zoonotic transmission of SARS-CoV might also be determined by species-specific variations in the ACE2 sequence. Thus, murine and rat ACE2, which do not (rat), or only inefficiently (murine), support SARS-S-driven entry (Li et al. 2004, 2005c), contain a leucine (human) to histidine (mouse, rat) exchange at position 353. This exchange impedes formation of robust contacts with T487 in SARS-S and thereby prevents murine and rat ACE2 from efficiently supporting SARS-S-driven cellular entry (Li et al. 2005a). In addition, the rat but not the murine receptor contains a M82N exchange, which introduces a glycosylation signal. The glycan added to N82 blocks the interaction with L472 in SARS-S and further decreases receptor function, explaining why rat ACE2 is less capable of supporting SARS-S-driven entry than murine ACE2 (Li et al. 2005a). These results, in conjunction with the aforementioned variations in the RBD sequence, highlight that the efficiency of the SARS-S interaction with ACE2 might be a critical determinant of interspecies transmission of SARS-CoV.

1.4.3 The Human Coronavirus NL63 Uses ACE2 for Cellular Entry

A novel human coronavirus, NL63, was discovered by two Dutch groups in the aftermath of the SARS-CoV outbreak (van der Hoek et al. 2004; Fouchier et al. 2004). NL63 is a group I CoV and shows high sequence similarity to the long-known human CoV 229E. The 229E virus, like all other group I viruses described at the time of the NL63 discovery, uses CD13 (aminopeptidase N) as a receptor for cellular entry (Hofmann and Pöhlmann 2004). Considering the specificity of group I viruses for CD13 and taking into account that the spike proteins of 229E and NL63 share 56% sequence identity (van der Hoek et al. 2004; Pyrc et al. 2004), it was surprising that NL63 was shown to use ACE2 and not CD13 for cellular entry (Hofmann et al. 2005). This finding raised the question of whether both viruses use similar strategies to engage ACE2. Mapping studies revealed that an N-terminal unique region in NL63-S, which was suspected to function as RBD, is in fact dispensable for receptor engagement (Hofmann et al. 2006). In contrast, several motifs within amino acids 232 and 684 were found to be required for ACE2 binding within an initial study, and it was suggested that NL63-S might not harbor a single continuous RBD (Hofmann et al. 2006). However, subsequent analyses narrowed the region responsible for ACE2 binding to amino acids 301–643 and 476–616, respectively, and a SARS-S RBM-like motif was identified in NL63-S (Li et al. 2007; Lin et al. 2008). Several amino acid substitutions in ACE2 were found to alter ACE2 usage by SARS-S but not by NL63-S (Hofmann et al. 2006), indicating that both S proteins might interact with different ACE2 surfaces. This interpretation is not undisputed (Li et al. 2007) and solution of the structure of NL63-S in complex with ACE2 might be required to clarify whether SARS-S and NL63-S recognize ACE2 differentially. In any case, it is clear that both viruses employ different mechanisms to activate membrane fusion once the S proteins have bound to ACE2. Thus, it is believed that upon ACE2 engagement SARS-CoV is internalized into endosomal vesicles, where the pH-dependent cellular protease cathepsin L activates SARS-S by cleavage (Simmons et al. 2005). In contrast, low pH and cathepsin activity seem to be largely dispensable for NL63-S-driven entry and it is at present unclear how NL63-S-driven membrane fusion is triggered (Huang et al. 2006; Hofmann et al. 2006).

1.4.4 SARS Versus NL63: A Correlation Between ACE2 Downregulation and Viral Pathogenicity?

NL63 is a globally distributed pathogen which is acquired early in childhood and does not usually cause severe disease (Pyrc et al. 2007). This observation contrasts with the high pathogenicity of SARS-CoV and raises the question of which viral factors determine disease severity. Again, S protein interactions with ACE2 might play a central role. Thus, a milestone discovery by Imai and colleagues indicated that ACE2 expression protects against development of acute respiratory distress syndrome (ARDS) (Imai et al. 2005). ACE2 is an integral component of the renin–angiotensin system (RAS), a key regulator of blood pressure and, as demonstrated by Imai and colleagues (Imai et al. 2005), lung function (Imai et al. 2008; Penninger et al. 2008). Knock-down of ACE2 in a mouse model caused accumulation of angiotensin II, which promoted development of ARDS by signaling via the AT1R receptor (Imai et al. 2005, 2008; Penninger et al. 2008). Conversely, inhibition of AT1R and application of soluble ACE2 protected against ARDS (Imai et al. 2005). Interestingly, a soluble form of the S1 subunit of SARS-S was shown to downregulate ACE2 expression in vitro and in vivo (Kuba et al. 2005), indicating that SARS-S engagement of ACE2 might promote SARS development even in the absence of productive infection. The S protein of NL63 exhibits a markedly reduced affinity for ACE2 compared to SARS-S (Mathewson et al. 2008) and seems to engage the receptor in a different fashion (Hofmann et al. 2006), suggesting that differential ACE2 downregulation by SARS-CoV and NL63 could contribute to the differential pathogenicity of these viruses. However, it is largely unclear how SARS-CoV decreases ACE2 expression and the effect of NL63 on ACE2 levels has not been systematically investigated. Notably, a recent study indicates that SARS-S might promote shedding of the ACE2 ectodomain by inducing ACE2 cleavage by TACE/ADAM17 (Fig. 1.2), a process that seems to be essential for infectious entry (Haga et al. 2008). In contrast, NL63-S did not induce appreciable ACE2 shedding (Haga et al. 2008). Thus, the previously observed ACE2 downregulation by SARS-S might have been due to proteolytic cleavage and dissociation of the ectodomain rather than ACE2 internalization and degradation. However, it is unclear if shedding of the ACE2 ectodomain actually exacerbates SARS development, considering that soluble ACE2 protects against ARDS in a mouse model (Imai et al. 2005).
Fig. 1.2

ACE2 downregulation by SARS-S might promote development of SARS (adapted from Kuba et al. 2006). ACE and ACE2 are key components of the renin–angiotensin system. ACE processes angiotensin I (ANG1) into angiotensin II (ANG2) and accumulation of ANG2 can promote acute lung failure via angiotensin II type 1 receptor (AT1R). This process is prevented by ACE2, which converts ANG2 into angiotensin 1-7 (ANG-(1-7)). The angiotensin II type 2 receptor (AT2R) also exerts a protective function. The interactions of SARS-S with ACE2 drive infectious entry but also induce downregulation of ACE2, possibly by promoting ACE2 cleavage by TACE/ADAM17. Diminished ACE2 expression then facilitates SARS development. ACE: angiotensin-converting enzyme; TACE: TNF-α converting enzyme; ADAM17: ADAM metallopeptidase domain 17

1.5 Cleavage by Endosomal Cathepsin Proteases Activates SARS-S

Class I fusion proteins usually require proteolytic cleavage to transit into an activated state (Hofmann and Pöhlmann 2004; Kielian and Rey 2006). However, the strategies to accomplish proteolytic activation can vary. Many fusion proteins are cleaved by subtilisin-like proteases in the secretory pathway of infected cells, and proteolytically processed proteins are incorporated into virions. This applies to the S proteins of most strains of murine hepatitis virus (MHV), a group II coronavirus. The membrane fusion reaction is subsequently triggered by binding of the cleaved S proteins to their cellular receptor, CEACAM-1 (Williams et al. 1991; Nash and Buchmeier 1997; de Haan et al. 2004; Qiu et al. 2006). Consequently, entry is pH-independent and encompasses fusion of the viral membrane with the plasma membrane of target cells (Nash and Buchmeier 1997; de Haan et al. 2004; Qiu et al. 2006). The influenza virus HA is either cleaved by subtilisin proteases in the secretory pathway or by secreted proteases present in the lung lumen. However, subsequent binding to the receptor determinant sialic acid does not trigger membrane fusion but internalization into endosomal vesicles, where fusion is triggered by low pH (Eckert and Kim 2001). Thus, infectious entry of influenza viruses is pH-dependent and is facilitated by fusion of the viral membrane with endosomal membranes (Eckert and Kim 2001).

The SARS-S protein employs a mixture of the entry strategies described above. At present, there is no evidence for appreciable cleavage of SARS-S produced in infected cells (Xiao et al. 2003; Yang et al. 2004; Simmons et al. 2004; Yao et al. 2004; Hofmann et al. 2004b), with the exception of a single report (Wu et al. 2004). It has been documented that the presence of furin can augment SARS-S activity and that a furin inhibitor blocks SARS-CoV infection (Bergeron et al. 2005; Follis et al. 2006). However, cleavage of SARS-S has not been detected under these conditions (Bergeron et al. 2005; Follis et al. 2006). Instead, a seminal study by Simmons and colleagues showed that SARS-S is activated by the endosomal, pH-dependent protease cathepsin L upon uptake into target cells, and that cathepsin L activity is essential for infectious entry (Simmons et al. 2005). Cathepsin B can also contribute to SARS-S activation but seems to be of minor importance compared to cathepsin L (Simmons et al. 2005). Importantly, appropriate SARS-S cleavage by cathepsin L seems to require a modest conformational rearrangement of SARS-S (Simmons et al. 2005), which is induced upon binding to ACE2 (Beniac et al. 2007). Thus, SARS-S-driven entry is pH-dependent and relies on fusion of viral and endosomal membranes (Yang et al. 2004; Simmons et al. 2004, 2005; Hofmann et al. 2004b). However, acidic conditions are required for cathepsin activity and have no triggering effect on SARS-S (Simmons et al. 2005).

The cathepsin L cleavage site in SARS-S was mapped to T678, when recombinant proteins were employed (Bosch et al. 2008), but evidence that T678 is important for SARS-CoV entry is lacking and cathepsin L-mediated cleavage of virion-associated SARS-S in target cells remains to be demonstrated. It is also unclear if cellular proteases other than cathepsin B and L can allow SARS-S-driven entry into certain target cells. An activating function of factor Xa has recently been suggested (Du et al. 2007) but the results await confirmation. Finally, it is noteworthy that engineered cleavage of SARS-S in virus-producing cells can ablate the need for cathepsin activity in target cells (Watanabe et al. 2008). This finding highlights the need to analyze if SARS-S is cleaved in primary lung cells and to determine if cathepsin activity is indeed required for viral spread in vivo – information pivotal to efforts aiming at the development of cathepsin inhibitors for antiviral therapy.

1.6 Membrane Fusion is Driven by Conserved Elements Located in the S2 Subunit of the SARS Spike Protein

The functional organization of SARS-S2 resembles that of the TMs of other class I fusion proteins and SARS-S-driven membrane fusion reaction follows the principles previously established for other class I fusion proteins (Hofmann and Pöhlmann 2004): membrane fusion commences by insertion of the fusion peptide into the target cell membrane (Fig. 1.3). In this context, it is worth noting that SARS-S, in contrast to, for example, HIV Env and influenza HA, contains an “internal” fusion peptide, which does not constitute the N terminus of S2 but may comprise amino acids 770–788 (Sainz et al. 2005). Upon fusion peptide insertion, the S2 subunit is connected with the viral and the target cell membrane. Subsequently, the C-terminal HR (termed HR2) folds back onto the N-terminal HR (termed HR1), forming an energetically stable six-helix bundle structure, in which HR1 and HR2 are oriented in an antiparallel fashion (Bosch et al. 2003; Tripet et al. 2004; Liu et al. 2004; Supekar et al. 2004; Ingallinella et al. 2004; Xu et al. 2004; Hsu et al. 2004). Thereby, viral and target cell membranes are pulled into close proximity, allowing the membranes to merge (Fig. 1.3). Peptides derived from HR2, which bind to HR1 and block the formation of the six-helix bundle, are used for therapy of HIV infection (Este and Telenti 2007). A similar approach was successful for blockade of SARS-CoV spread in cell culture (Bosch et al. 2003; Liu et al. 2004; Zhu et al. 2004; Yuan et al. 2004; Ni et al. 2005), but the inhibitors developed were not as potent as those used to treat HIV infection. One reason for the decreased potency might be inherent to the cellular location of the membrane fusion reaction: the HIV Env protein drives fusion with the plasma cell membrane, and the target of the inhibitory peptides is readily accessible. In contrast, SARS-CoV fuses with endosomal membranes, and inhibitors must be taken up into endosomes to efficiently block the fusion reaction. Potentially, this could present a significant hurdle to the development of fusion inhibitors for therapy of SARS-CoV infection (Watanabe et al. 2008).
Fig. 1.3

Cellular entry of SARS-CoV and its inhibition (adapted from Hofmann and Pöhlmann 2004). The cellular entry of SARS-CoV commences by binding of the S protein to its receptor ACE2. Bound virus is then taken up into target cells, possibly by a clathrin- and caveolae-independent mechanism (Wang et al. 2008). The S protein is cleaved by the pH-dependent cellular protease cathepsin L in endosomes, and cathepsin L activity is essential for infectious entry. The membrane fusion reaction starts with the insertion of the fusion peptide into the target cell membrane. Formation of the stable six-helix bundle structure brings the viral and the target cell membrane into close proximity and is intimately associated with membrane fusion. The fusion reaction can be inhibited by HR2-derived peptides, which bind into a groove on HR1 and thereby prevent back-folding of HR2 onto HR1 and thus the formation of the six-helix bundle structure

1.7 Conclusions

The cellular entry of SARS-CoV is a multistep process which involves the formation of several transient intermediates. All structures participating in the entry cascade are potential targets for inhibitors and the feasibility of several approaches to prevent entry has already been demonstrated. The first step, SARS-S engagement of ACE2, is an attractive target for both preventive and therapeutic approaches. Thus, the immunization with the RBD has been shown to elicit neutralizing antibodies (He et al. 2004a, 2005), and monoclonal RBD-specific antibodies which exhibit potent antiviral effects in animal models have been identified (Sui et al. 2004, 2005; Rockx et al. 2007; Zhu et al. 2007). In addition, SARS-S binding to ACE2 can be inhibited by nonpeptidic molecules targeting the receptor (Huentelman et al. 2004), and soluble ACE2 was shown to block SARS-CoV infection (Hofmann et al. 2004a) and to protect against ARDS (Kuba et al. 2005), making this approach particularly promising. The S protein can also be targeted by lectins, which bind glycans on the S protein and thereby block viral entry (van der Meer et al. 2007; Keyaerts et al. 2007), albeit issues concerning potential toxicity and antigenicity remain to be addressed. After binding to ACE2 and uptake into target cells, the S protein must be activated by cathepsin L, and potent cathepsin L inhibitors are available (Simmons et al. 2005). However, the role of cathepsin L in viral spread in vivo remains to be assessed, and knock-out mice (Reinheckel et al. 2001) might be suitable tools for these endeavours. Finally, inhibitors of the fusion reaction have been described (Bosch et al. 2003; Liu et al. 2004; Zhu et al. 2004; Yuan et al. 2004; Ni et al. 2005), but optimization of available compounds and generation of nonpeptidic compounds is desirable. In summary, the approaches described above, particularly combinations thereof, should allow development of compounds suitable for effectively preventing or combating future outbreaks of SARS-CoV.



We thank T.F. Schulz for support and BMBF for funding.


  1. Babcock GJ, Esshaki DJ, Thomas WD Jr, Ambrosino DM (2004) Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol 78:4552–4560PubMedGoogle Scholar
  2. Bashirova AA, Geijtenbeek TB, van Duijnhoven GC, van Vliet SJ, Eilering JB, Martin MP, Wu L, Martin TD, Viebig N, Knolle PA, Kewalramani VN, van Kooyk Y, Carrington M (2001) A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 193:671–678PubMedGoogle Scholar
  3. Beniac DR, deVarennes SL, Andonov A, He R, Booth TF (2007) Conformational reorganization of the SARS coronavirus spike following receptor binding: implications for membrane fusion. PLoS ONE 2:e1082PubMedGoogle Scholar
  4. Bergeron E, Vincent MJ, Wickham L, Hamelin J, Basak A, Nichol ST, Chretien M, Seidah NG (2005) Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus. Biochem Biophys Res Commun 326:554–563PubMedGoogle Scholar
  5. Bosch BJ, van der Zee R, de Haan CA, Rottier PJ (2003) The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 77:8801–8811PubMedGoogle Scholar
  6. Bosch BJ, Bartelink W, Rottier PJ (2008) Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the fusion peptide. J Virol 82:8887–8890PubMedGoogle Scholar
  7. Chan VS, Chan KY, Chen Y, Poon LL, Cheung AN, Zheng B, Chan KH, Mak W, Ngan HY, Xu X, Screaton G, Tam PK, Austyn JM, Chan LC, Yip SP, Peiris M, Khoo US, Lin CL (2006) Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection. Nat Genet 38:38–46PubMedGoogle Scholar
  8. de Haan CA, Stadler K, Godeke GJ, Bosch BJ, Rottier PJ (2004) Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell−cell but not virus−cell fusion. J Virol 78:6048–6054PubMedGoogle Scholar
  9. Ding Y, He L, Zhang Q, Huang Z, Che X, Hou J, Wang H, Shen H, Qiu L, Li Z, Geng J, Cai J, Han H, Li X, Kang W, Weng D, Liang P, Jiang S (2004) Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol 203:622–630PubMedGoogle Scholar
  10. Drosten C, Gunther S, Preiser W, van der WS, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976PubMedGoogle Scholar
  11. Du L, Kao RY, Zhou Y, He Y, Zhao G, Wong C, Jiang S, Yuen KY, Jin DY, Zheng BJ (2007) Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity. Biochem Biophys Res Commun 359:174–179PubMedGoogle Scholar
  12. Eckert DM, Kim PS (2001) Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem 70:777–810PubMedGoogle Scholar
  13. Este JA, Telenti A (2007) HIV entry inhibitors. Lancet 370:81–88PubMedGoogle Scholar
  14. Follis KE, York J, Nunberg JH (2006) Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell−cell fusion but does not affect virion entry. Virology 350:358–369PubMedGoogle Scholar
  15. Fouchier RA, Hartwig NG, Bestebroer TM, Niemeyer B, de Jong JC, Simon JH, Osterhaus AD (2004) A previously undescribed coronavirus associated with respiratory disease in humans. Proc Natl Acad Sci USA 101:6212–6216PubMedGoogle Scholar
  16. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, Kewalramani VN, Littman DR, Figdor CG, van Kooyk Y (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100:587–597PubMedGoogle Scholar
  17. Gramberg T, Hofmann H, Moller P, Lalor PF, Marzi A, Geier M, Krumbiegel M, Winkler T, Kirchhoff F, Adams DH, Becker S, Münch J, Pöhlmann S (2005) LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus. Virology 340:224–236PubMedGoogle Scholar
  18. Gu J, Korteweg C (2007) Pathology and pathogenesis of severe acute respiratory syndrome. Am J Pathol 170:1136–1147PubMedGoogle Scholar
  19. Gu J, Gong E, Zhang B, Zheng J, Gao Z, Zhong Y, Zou W, Zhan J, Wang S, Xie Z, Zhuang H, Wu B, Zhong H, Shao H, Fang W, Gao D, Pei F, Li X, He Z, Xu D, Shi X, Anderson VM, Leong AS (2005) Multiple organ infection and the pathogenesis of SARS. J Exp Med 202:415–424PubMedGoogle Scholar
  20. Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, Cheung CL, Luo SW, Li PH, Zhang LJ, Guan YJ, Butt KM, Wong KL, Chan KW, Lim W, Shortridge KF, Yuen KY, Peiris JS, Poon LL (2003) Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302:276–278PubMedGoogle Scholar
  21. Haga S, Yamamoto N, Nakai-Murakami C, Osawa Y, Tokunaga K, Sata T, Yamamoto N, Sasazuki T, Ishizaka Y (2008) Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry. Proc Natl Acad Sci USA 105:7809–7814PubMedGoogle Scholar
  22. Hamming I, Timens W, Bulthuis ML, Lely AT, Navis GJ, van Goor H (2004) Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 203:631–637PubMedGoogle Scholar
  23. Han DP, Lohani M, Cho MW (2007) Specific asparagine-linked glycosylation sites are critical for DC-SIGN- and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry. J Virol 81:12029–12039PubMedGoogle Scholar
  24. He Y, Zhou Y, Liu S, Kou Z, Li W, Farzan M, Jiang S (2004a) Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine. Biochem Biophys Res Commun 324:773–781PubMedGoogle Scholar
  25. He Y, Zhou Y, Wu H, Luo B, Chen J, Li W, Jiang S (2004b) Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines. J Immunol 173:4050–4057PubMedGoogle Scholar
  26. He Y, Lu H, Siddiqui P, Zhou Y, Jiang S (2005) Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies. J Immunol 174:4908–4915PubMedGoogle Scholar
  27. Hofmann H, Pöhlmann S (2004) Cellular entry of the SARS coronavirus. Trends Microbiol 12:466–472PubMedGoogle Scholar
  28. Hofmann H, Geier M, Marzi A, Krumbiegel M, Peipp M, Fey GH, Gramberg T, Pöhlmann S (2004a) Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor. Biochem Biophys Res Commun 319:1216–1221PubMedGoogle Scholar
  29. Hofmann H, Hattermann K, Marzi A, Gramberg T, Geier M, Krumbiegel M, Kuate S, Uberla K, Niedrig M, Pöhlmann S (2004b) S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J Virol 78:6134–6142PubMedGoogle Scholar
  30. Hofmann H, Pyrc K, van der HL, Geier M, Berkhout B, Pöhlmann S (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc Natl Acad Sci USA 102:7988–7993PubMedGoogle Scholar
  31. Hofmann H, Simmons G, Rennekamp AJ, Chaipan C, Gramberg T, Heck E, Geier M, Wegele A, Marzi A, Bates P, Pöhlmann S (2006) Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors. J Virol 80:8639–8652PubMedGoogle Scholar
  32. Hsieh PK, Chang SC, Huang CC, Lee TT, Hsiao CW, Kou YH, Chen IY, Chang CK, Huang TH, Chang MF (2005) Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent. J Virol 79:13848–13855PubMedGoogle Scholar
  33. Hsu CH, Ko TP, Yu HM, Tang TK, Chen ST, Wang AH (2004) Immunological, structural, and preliminary X-ray diffraction characterizations of the fusion core of the SARS-coronavirus spike protein. Biochem Biophys Res Commun 324:761–767PubMedGoogle Scholar
  34. Huang Y, Yang ZY, Kong WP, Nabel GJ (2004) Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production. J Virol 78:12557–12565PubMedGoogle Scholar
  35. Huang IC, Bosch BJ, Li F, Li W, Lee KH, Ghiran S, Vasilieva N, Dermody TS, Harrison SC, Dormitzer PR, Farzan M, Rottier PJ, Choe H (2006) SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells. J Biol Chem 281:3198–3203PubMedGoogle Scholar
  36. Huentelman MJ, Zubcevic J, Hernandez Prada JA, Xiao X, Dimitrov DS, Raizada MK, Ostrov DA (2004) Structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor. Hypertension 44:903–906PubMedGoogle Scholar
  37. Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, Crackower MA, Fukamizu A, Hui CC, Hein L, Uhlig S, Slutsky AS, Jiang C, Penninger JM (2005) Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 436:112–116PubMedGoogle Scholar
  38. Imai Y, Kuba K, Penninger JM (2008) The discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice. Exp Physiol 93:543–548PubMedGoogle Scholar
  39. Ingallinella P, Bianchi E, Finotto M, Cantoni G, Eckert DM, Supekar VM, Bruckmann C, Carfi A, Pessi A (2004) Structural characterization of the fusion-active complex of severe acute respiratory syndrome (SARS) coronavirus. Proc Natl Acad Sci USA 101:8709–8714PubMedGoogle Scholar
  40. Jeffers SA, Tusell SM, Gillim-Ross L, Hemmila EM, Achenbach JE, Babcock GJ, Thomas WD Jr, Thackray LB, Young MD, Mason RJ, Ambrosino DM, Wentworth DE, Demartini JC, Holmes KV (2004) CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci USA 101:15748–15753PubMedGoogle Scholar
  41. Keyaerts E, Vijgen L, Pannecouque C, Van Damme E, Peumans W, Egberink H, Balzarini J, Van Ranst M (2007) Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle. Antivir Res 75:179–187PubMedGoogle Scholar
  42. Khoo US, Chan KY, Chan VS, Lin CL (2008) DC-SIGN and L-SIGN: the SIGNs for infection. J Mol Med 86:861–874PubMedGoogle Scholar
  43. Kielian M (2006) Class II virus membrane fusion proteins. Virology 344:38–47PubMedGoogle Scholar
  44. Kielian M, Rey FA (2006) Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol 4:67–76PubMedGoogle Scholar
  45. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ (2003) A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966PubMedGoogle Scholar
  46. Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, Huan Y, Yang P, Zhang Y, Deng W, Bao L, Zhang B, Liu G, Wang Z, Chappell M, Liu Y, Zheng D, Leibbrandt A, Wada T, Slutsky AS, Liu D, Qin C, Jiang C, Penninger JM (2005) A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 11:875–879PubMedGoogle Scholar
  47. Kuba K, Imai Y, Rao S, Jiang C, Penninger JM (2006) Lessons from SARS: control of acute lung failure by the SARS receptor ACE2. J Mol Med 84:814–820PubMedGoogle Scholar
  48. Lanoue A, Clatworthy MR, Smith P, Green S, Townsend MJ, Jolin HE, Smith KG, Fallon PG, McKenzie AN (2004) SIGN-R1 contributes to protection against lethal pneumococcal infection in mice. J Exp Med 200:1383–1393PubMedGoogle Scholar
  49. Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, Wong SS, Leung SY, Chan KH, Yuen KY (2005) Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci USA 102:14040–14045PubMedGoogle Scholar
  50. Li F (2008) Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections. J Virol 82:6984–6991PubMedGoogle Scholar
  51. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450–454PubMedGoogle Scholar
  52. Li W, Greenough TC, Moore MJ, Vasilieva N, Somasundaran M, Sullivan JL, Farzan M, Choe H (2004) Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2. J Virol 78:11429–11433PubMedGoogle Scholar
  53. Li F, Li W, Farzan M, Harrison SC (2005a) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor I2005. Science 309:1864–1868PubMedGoogle Scholar
  54. Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, Wang H, Crameri G, Hu Z, Zhang H, Zhang J, McEachern J, Field H, Daszak P, Eaton BT, Zhang S, Wang LF (2005b) Bats are natural reservoirs of SARS-like coronaviruses. Science 310:676–679PubMedGoogle Scholar
  55. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, Wong SK, Huang IC, Xu K, Vasilieva N, Murakami A, He Y, Marasco WA, Guan Y, Choe H, Farzan M (2005c) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24:1634–1643PubMedGoogle Scholar
  56. Li W, Sui J, Huang IC, Kuhn JH, Radoshitzky SR, Marasco WA, Choe H, Farzan M (2007) The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2. Virology 367:367–374PubMedGoogle Scholar
  57. Lin HX, Feng Y, Wong G, Wang L, Li B, Zhao X, Li Y, Smaill F, Zhang C (2008) Identification of residues in the receptor-binding domain (RBD) of the spike protein of human coronavirus NL63 that are critical for the RBD-ACE2 receptor interaction. J Gen Virol 89:1015–1024PubMedGoogle Scholar
  58. Liu S, Xiao G, Chen Y, He Y, Niu J, Escalante CR, Xiong H, Farmar J, Debnath AK, Tien P, Jiang S (2004) Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 363:938–947PubMedGoogle Scholar
  59. Marzi A, Gramberg T, Simmons G, Moller P, Rennekamp AJ, Krumbiegel M, Geier M, Eisemann J, Turza N, Saunier B, Steinkasserer A, Becker S, Bates P, Hofmann H, Pöhlmann S (2004) DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome Coronavirus. J Virol 78:12090–12095PubMedGoogle Scholar
  60. Mathewson AC, Bishop A, Yao Y, Kemp F, Ren J, Chen H, Xu X, Berkhout B, van der HL, Jones IM (2008) Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2. J Gen Virol 89:2741–2745PubMedGoogle Scholar
  61. McBride CE, Li J, Machamer CE (2007) The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein. J Virol 81:2418–2428PubMedGoogle Scholar
  62. Mossel EC, Huang C, Narayanan K, Makino S, Tesh RB, Peters CJ (2005) Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication. J Virol 79:3846–3850PubMedGoogle Scholar
  63. Mossel EC, Wang J, Jeffers S, Edeen KE, Wang S, Cosgrove GP, Funk CJ, Manzer R, Miura TA, Pearson LD, Holmes KV, Mason RJ (2008) SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells. Virology 372:127–135PubMedGoogle Scholar
  64. Nal B, Chan C, Kien F, Siu L, Tse J, Chu K, Kam J, Staropoli I, Crescenzo-Chaigne B, Escriou N, van der WS, Yuen KY, Altmeyer R (2005) Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E. J Gen Virol 86:1423–1434PubMedGoogle Scholar
  65. Nash TC, Buchmeier MJ (1997) Entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways. Virology 233:1–8PubMedGoogle Scholar
  66. Ni L, Zhu J, Zhang J, Yan M, Gao GF, Tien P (2005) Design of recombinant protein-based SARS-CoV entry inhibitors targeting the heptad-repeat regions of the spike protein S2 domain. Biochem Biophys Res Commun 330:39–45PubMedGoogle Scholar
  67. Nie Y, Wang P, Shi X, Wang G, Chen J, Zheng A, Wang W, Wang Z, Qu X, Luo M, Tan L, Song X, Yin X, Chen J, Ding M, Deng H (2004) Highly infectious SARS-CoV pseudotyped virus reveals the cell tropism and its correlation with receptor expression. Biochem Biophys Res Commun 321:994–1000PubMedGoogle Scholar
  68. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, Nicholls J, Yee WK, Yan WW, Cheung MT, Cheng VC, Chan KH, Tsang DN, Yung RW, Ng TK, Yuen KY (2003a) Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319–1325PubMedGoogle Scholar
  69. Peiris JS, Yuen KY, Osterhaus AD, Stohr K (2003b) The severe acute respiratory syndrome. N Engl J Med 349:2431–2441PubMedGoogle Scholar
  70. Peiris JS, Guan Y, Yuen KY (2004) Severe acute respiratory syndrome. Nat Med 10:S88–S97PubMedGoogle Scholar
  71. Penninger J, Imai Y, Kuba K (2008) The discovery of ACE2 and its role in acute lung injury. Exp Physiol 93:543–548Google Scholar
  72. Pöhlmann S, Soilleux EJ, Baribaud F, Leslie GJ, Morris LS, Trowsdale J, Lee B, Coleman N, Doms RW (2001) DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci USA 98:2670–2675PubMedGoogle Scholar
  73. Pyrc K, Jebbink MF, Berkhout B, van der HL (2004) Genome structure and transcriptional regulation of human coronavirus NL63. Virol J 1:7PubMedGoogle Scholar
  74. Pyrc K, Berkhout B, van der HL (2007) Identification of new human coronaviruses. Expert Rev Anti Infect Ther 5:245–253PubMedGoogle Scholar
  75. Qiu Z, Hingley ST, Simmons G, Yu C, Das SJ, Bates P, Weiss SR (2006) Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry. J Virol 80:5768–5776PubMedGoogle Scholar
  76. Reinheckel T, Deussing J, Roth W, Peters C (2001) Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L. Biol Chem 382:735–741PubMedGoogle Scholar
  77. Ren W, Qu X, Li W, Han Z, Yu M, Zhou P, Zhang SY, Wang LF, Deng H, Shi Z (2008) Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin. J Virol 82:1899–1907PubMedGoogle Scholar
  78. Rockx B, Sheahan T, Donaldson E, Harkema J, Sims A, Heise M, Pickles R, Cameron M, Kelvin D, Baric R (2007) Synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice. J Virol 81:7410–7423PubMedGoogle Scholar
  79. Sainz B Jr, Rausch JM, Gallaher WR, Garry RF, Wimley WC (2005) Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein. J Virol 79:7195–7206PubMedGoogle Scholar
  80. Schaefer M, Reiling N, Fessler C, Stephani J, Taniuchi I, Hatam F, Yildirim AO, Fehrenbach H, Walter K, Ruland J, Wagner H, Ehlers S, Sparwasser T (2008) Decreased pathology and prolonged survival of human DC-SIGN transgenic mice during mycobacterial infection. J Immunol 180:6836–6845PubMedGoogle Scholar
  81. Shih YP, Chen CY, Liu SJ, Chen KH, Lee YM, Chao YC, Chen YM (2006) Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus. J Virol 80:10315–10324PubMedGoogle Scholar
  82. Simmons G, Reeves JD, Rennekamp AJ, Amberg SM, Piefer AJ, Bates P (2004) Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc Natl Acad Sci USA 101:4240–4245PubMedGoogle Scholar
  83. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, Bates P (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci USA 102:11876–11881PubMedGoogle Scholar
  84. Sims AC, Burkett SE, Yount B, Pickles RJ (2008) SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium. Virus Res 133:33–44PubMedGoogle Scholar
  85. Siu YL, Teoh KT, Lo J, Chan CM, Kien F, Escriou N, Tsao SW, Nicholls JM, Altmeyer R, Peiris JS, Bruzzone R, Nal B (2008) The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles. J Virol 82:11318–11330PubMedGoogle Scholar
  86. Song HD, Tu CC, Zhang GW, Wang SY, Zheng K, Lei LC, Chen QX, Gao YW, Zhou HQ, Xiang H, Zheng HJ, Chern SW, Cheng F, Pan CM, Xuan H, Chen SJ, Luo HM, Zhou DH, Liu YF, He JF, Qin PZ, Li LH, Ren YQ, Liang WJ, Yu YD, Anderson L, Wang M, Xu RH, Wu XW, Zheng HY, Chen JD, Liang G, Gao Y, Liao M, Fang L, Jiang LY, Li H, Chen F, Di B, He LJ, Lin JY, Tong S, Kong X, Du L, Hao P, Tang H, Bernini A, Yu XJ, Spiga O, Guo ZM, Pan HY, He WZ, Manuguerra JC, Fontanet A, Danchin A, Niccolai N, Li YX, Wu CI, Zhao GP (2005) Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc Natl Acad Sci USA 102:2430–2435PubMedGoogle Scholar
  87. Stadler K, Rappuoli R (2005) SARS: understanding the virus and development of rational therapy. Curr Mol Med 5:677–697PubMedGoogle Scholar
  88. Stadler K, Masignani V, Eickmann M, Becker S, Abrignani S, Klenk HD, Rappuoli R (2003) SARS–beginning to understand a new virus. Nat Rev Microbiol 1:209–218PubMedGoogle Scholar
  89. Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L, Garcia-Sastre A, Weber F, Kochs G (2007) The intracellular sites of early replication and budding of SARS-coronavirus. Virology 361:304–315PubMedGoogle Scholar
  90. Sui J, Li W, Murakami A, Tamin A, Matthews LJ, Wong SK, Moore MJ, Tallarico AS, Olurinde M, Choe H, Anderson LJ, Bellini WJ, Farzan M, Marasco WA (2004) Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA 101:2536–2541PubMedGoogle Scholar
  91. Sui J, Li W, Roberts A, Matthews LJ, Murakami A, Vogel L, Wong SK, Subbarao K, Farzan M, Marasco WA (2005) Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants. J Virol 79:5900–5906PubMedGoogle Scholar
  92. Supekar VM, Bruckmann C, Ingallinella P, Bianchi E, Pessi A, Carfi A (2004) Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein. Proc Natl Acad Sci USA 101:17958–17963PubMedGoogle Scholar
  93. Tang NL, Chan PK, HDS To KF, Zhang W, Chan FK, Sung JJ, Lo YM (2007) Lack of support for an association between CLEC4M homozygosity and protection against SARS coronavirus infection. Nat Genet 39:691–692PubMedGoogle Scholar
  94. To KF, Lo AW (2004) Exploring the pathogenesis of severe acute respiratory syndrome (SARS): the tissue distribution of the coronavirus (SARS-CoV) and its putative receptor, angiotensin-converting enzyme 2 (ACE2). J Pathol 203:740–743PubMedGoogle Scholar
  95. Towler P, Staker B, Prasad SG, Menon S, Tang J, Parsons T, Ryan D, Fisher M, Williams D, Dales NA, Patane MA, Pantoliano MW (2004) ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis. J Biol Chem 279:17996–18007PubMedGoogle Scholar
  96. Tripet B, Howard MW, Jobling M, Holmes RK, Holmes KV, Hodges RS (2004) Structural characterization of the SARS-coronavirus spike S fusion protein core. J Biol Chem 279:20836–20849PubMedGoogle Scholar
  97. van der Hoek L, Pyrc K, Jebbink MF, Vermeulen-Oost W, Berkhout RJ, Wolthers KC, Wertheim-van Dillen PM, Kaandorp J, Spaargaren J, Berkhout B (2004) Identification of a new human coronavirus. Nat Med 10:368–373PubMedGoogle Scholar
  98. van der Meer FJ, de Haan CA, Schuurman NM, Haijema BJ, Peumans WJ, Van Damme EJ, Delputte PL, Balzarini J, Egberink HF (2007) Antiviral activity of carbohydrate-binding agents against Nidovirales in cell culture. Antivir Res 76:21–29PubMedGoogle Scholar
  99. Wang P, Chen J, Zheng A, Nie Y, Shi X, Wang W, Wang G, Luo M, Liu H, Tan L, Song X, Wang Z, Yin X, Qu X, Wang X, Qing T, Ding M, Deng H (2004) Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun 315:439–444PubMedGoogle Scholar
  100. Wang H, Yang P, Liu K, Guo F, Zhang Y, Zhang G, Jiang C (2008) SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 18:290–301PubMedGoogle Scholar
  101. Watanabe R, Matsuyama S, Shirato K, Maejima M, Fukushi S, Morikawa S, Taguchi F (2008) Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved s protein as revealed by pseudotype virus bearing cleaved s protein. J Virol 82:11985–11991PubMedGoogle Scholar
  102. Williams RK, Jiang GS, Holmes KV (1991) Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci USA 88:5533–5536PubMedGoogle Scholar
  103. Wong SK, Li W, Moore MJ, Choe H, Farzan M (2004) A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279:3197–3201PubMedGoogle Scholar
  104. Wu XD, Shang B, Yang RF, Yu H, Ma ZH, Shen X, Ji YY, Lin Y, Wu YD, Lin GM, Tian L, Gan XQ, Yang S, Jiang WH, Dai EH, Wang XY, Jiang HL, Xie YH, Zhu XL, Pei G, Li L, Wu JR, Sun B (2004) The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells. Cell Res 14:400–406PubMedGoogle Scholar
  105. Xiao X, Chakraborti S, Dimitrov AS, Gramatikoff K, Dimitrov DS (2003) The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun 312:1159–1164PubMedGoogle Scholar
  106. Xu Y, Lou Z, Liu Y, Pang H, Tien P, Gao GF, Rao Z (2004) Crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem 279:49414–49419PubMedGoogle Scholar
  107. Yang ZY, Huang Y, Ganesh L, Leung K, Kong WP, Schwartz O, Subbarao K, Nabel GJ (2004) pH-Dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol 78:5642–5650PubMedGoogle Scholar
  108. Yao YX, Ren J, Heinen P, Zambon M, Jones IM (2004) Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein. J Infect Dis 190:91–98PubMedGoogle Scholar
  109. Yen YT, Liao F, Hsiao CH, Kao CL, Chen YC, Wu-Hsieh BA (2006) Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro. J Virol 80:2684–2693PubMedGoogle Scholar
  110. Yuan K, Yi L, Chen J, Qu X, Qing T, Rao X, Jiang P, Hu J, Xiong Z, Nie Y, Shi X, Wang W, Ling C, Yin X, Fan K, Lai L, Ding M, Deng H (2004) Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein. Biochem Biophys Res Commun 319:746–752PubMedGoogle Scholar
  111. Zhi L, Zhou G, Zhang H, Zhai Y, Yang H, Zhang F, Wang S, Wei M, He F (2007) Lack of support for an association between CLEC4M homozygosity and protection against SARS coronavirus infection. Nat Genet 39:692–694PubMedGoogle Scholar
  112. Zhu J, Xiao G, Xu Y, Yuan F, Zheng C, Liu Y, Yan H, Cole DK, Bell JI, Rao Z, Tien P, Gao GF (2004) Following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors. Biochem Biophys Res Commun 319:283–288PubMedGoogle Scholar
  113. Zhu Z, Chakraborti S, He Y, Roberts A, Sheahan T, Xiao X, Hensley LE, Prabakaran P, Rockx B, Sidorov IA, Corti D, Vogel L, Feng Y, Kim JO, Wang LF, Baric R, Lanzavecchia A, Curtis KM, Nabel GJ, Subbarao K, Jiang S, Dimitrov DS (2007) Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. Proc Natl Acad Sci USA 104:12123–12128PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Ilona Glowacka
  • Stephanie Bertram
  • Stefan Pöhlmann
    • 1
    Email author
  1. 1.Institute of Virology, OE 5230Hannover Medical SchoolHannoverGermany

Personalised recommendations