Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis

3.2.1. Trypsin

Trypsin (Fig. 2B) is a prototype serine endopeptidase and has been studied extensively in the context of virus glycoprotein cleavage-activation. Trypsin cleaves at neutral pH, with an optimum of approximately 8.0. Biochemically, trypsin strongly prefers to cleave at arginine (R) or lysine (K) residues, although almost all viral substrates get cleaved at arginines. Other flanking residues are in general non-discriminatory, however basic or hydrophobic residues at P2 decreases the catalytic activity, as does a proline (P) residue at P3 (Halfon et al., 2004). Overall the requirement for cleavage is for a single basic residue at P1. As trypsin is not very selective in terms of substrate recognition, there are potentially many sites within coronavirus S proteins that may be cleaved by it. However, within the native trimeric conformation of S, only a few of these sites, if any, are accessible to trypsin cleavage. Trypsin is expressed in the respiratory tract, where its activity is inhibited by alpha-1 antitrypsin. Trypsin is principally a digestive enzyme, with active trypsin found in the small intestine; as such trypsin is likely to be able to directly cleave the S proteins of many enteric coronaviruses. Probably the best example of this is porcine epidemic diarrhea virus (PEDV), where most strains are highly trypsin-dependent. In the case of PEDV produced in Vero cells, trypsin cleavage only occurs after receptor binding, and not on free particles (Park et al., 2011a). For those coronaviruses with respiratory tissue tropism, cell-culture based studies using trypsin likely act as a surrogate for more biologically relevant proteases such as members of the TMPRSS family, which have similar substrate specificities. A good example of this is SARS-CoV produced in VeroE6 cells where trypsin can override the need for cathepsin-mediated cleavage, and shifts the virus to a low-pH independent route of entry, possibly at the plasma membrane. As with PEDV, prior binding to the viral receptor is a pre-requisite for SARS-CoV fusion activation (Matsuyama et al., 2005). In other cases, such as with feline coronavirus (FCoV), trypsin can activate the virus for fusion without the need for prior engagement with the viral receptor (Costello et al., 2013).

3.2.2. Furin and the proprotein convertase (PC) family

A large number of coronaviruses, from different genera, possess at their S1/S2 boundary site a furin cleavage site (Table 1). Furin (Fig. 2B) and furin-like proteases cleave at paired basic residues, and belong to the group of proteases called proprotein convertases.

The PC family is comprised of nine serine proteases that can cleave a vast number of cellular and microbial protein substrates. Furin belongs to the subset of PCs that includes proprotein convertase 1 (PC1), PC2, PC4, PC5, paired basic amino acid cleaving enzyme 4 (PACE4) and PC7, which generally cleave at single or paired basic residues within the following motif R/K-(X)_0,2,4,6-R/K (X: any amino acid) (Seidah and Prat, 2012). The other two PCs, subtilisin/kexin-isozyme-1 (SKI-1) and proprotein convertase subtilisin/kexin type 9 (PCSK9) cleave at non-basic residue sites. SKI-1 recognizes R-X(-L/V/I)-X motifs (L: leucine, V: valine, I: isoleucine) and PCSK9 only autoproteolytically cleaves itself at the V–F–A–Q motif (F: phenylalanine, A: alanine, Q: glutamine). PCs are crucial regulatory proteases as they can activate and sometimes inactivate a very wide variety of substrates, such as enzymes and other proteases, cell adhesion factors, growth factors and hormones. The critical role of PCs is further demonstrated by knockout mouse experimental models, where individual knockout of furin, PC5, PACE4 or SKI-1 are lethal at different stages of development (Seidah and Prat, 2012).

PCs are known to be subverted by pathogenic microorganisms as they are recruited by viruses to process viral proteins, such as HIV gp160, respiratory syncytial virus (RSV) F protein, human papilloma virus minor capsid protein L2, dengue virus prM, and Chikungunya virus E3E2 (McCune et al., 1988, Ozden et al., 2008, Richards et al., 2006, Rodenhuis-Zybert et al., 2010, Zimmer et al., 2001). They are also implicated in the cleavage of bacterial toxins, e.g. Pseudomonas exotoxin and Shiga toxin (Molloy and Thomas, 2001).

Because furin can be viewed as a representative PC (Thomas, 2002), and because it is the most frequently characterized PC to be involved in processing and activating coronavirus S proteins, we will focus on this protease. However, it is important to recognize that there is a degree of redundancy between substrate recognition motifs by the different PCs, especially between furin, PC5 and PACE4. In line with this, a study has shown that overexpression of PACE4 and PC5 could enhance proteolytic cleavage of SARS-CoV S, along with furin (Bergeron et al., 2005).

Like all PCs, furin is a serine protease, containing a critical serine residue in its catalytic triad. Also, like all PCs, it is related to the bacterial subtilisin family of protease, in which the prototypical yeast protease kexin is found. PCs require Ca²⁺ ions to be active. While most PCs, including furin require neutral pH for activity, a study has shown that self-activation of furin via protonation of a regulatory histidine residue (His-69) requires a slightly acidic environment (Williamson et al., 2013). The minimal substrate recognition motif for furin is R-X-X-R, with the R-X-R/K-R motif being highly favorable. The fairly strict requirement for basic arginines at P1 and P4 positions as well as lysine at P2 is due to furin's binding pocket containing complementary charged residues (Henrich et al., 2003). Also, flanking serine (S) residues in the vicinity of the cleavage site appear to be highly favored by furin.

Several computational tools, such as ProP 1.0 and PiTou 2.0, have been made available for users to query amino acid sequences and predict whether or not they can be cleaved by furin (Duckert et al., 2004, Tian et al., 2012). The PiTou 2.0 tool offers the advantage of analyzing the 20 residue recognition motif used by furin (Tian, 2009, Tian et al., 2011), and takes into account the binding strength and solvent accessibility of substrate residues, allowing for high sensitivity and specificity predictions (Tian et al., 2012).

Furin is widely expressed and is often considered as a ubiquitous protease. However it should be noted that in most cases, the levels of furin expression are low (Shapiro et al., 1997). Furin is produced in the ER, and traffics along the secretory pathway, where it can process coronavirus S proteins in infected cells. Furin is a membrane bound protease that can be shed in the extracellular space, is constitutively secreted, and is mainly found in the trans-golgi network (TGN) where it is activated and can traffic to the plasma membrane. Furin can also recycle back from the plasma membrane to the TGN through an endocytic pathway (Seidah and Prat, 2012).

It is noteworthy that while most coronaviruses that are cleaved by furin or furin-like proteases are cleaved at only one site, the S1/S2 site, there are examples of coronaviruses, such as IBV and MERS-CoV that can be additionally cleaved at the putative fusion peptide-proximal site S2′ (Table 2), as described in Section 2.4 Proteolytic activation mechanisms of coronaviruses.

3.2.3. Cathepsins

The cathepsins comprise a group of cysteine, serine, and aspartyl proteases with both endo- and exo-peptidase activity that are typically found in endosomes and lysosomes and function as degradative enzymes, as well as in antigen processing.

Cathepsins may also be secreted however, especially in cancer cells (Mohamed and Sloane, 2006). As a general rule, the substrate specificity of cathepsins is quite broad, and analysis of the residues recognized by these enzymes seems to differ depending on the analyses used. Biochemical analysis of peptide substrates indicates that arginine (R) residues are preferred at the P1 position, often with an aromatic residue at P2 (Choe et al., 2006, Polgár, 1989, Rawlings and Barrett, 2004). However, databases of natural cathepsin substrates (e.g. MEROPS) show a preference for non-polar residues such as glycine (G), alanine (A), serine (S), threonine (T), and glutamine (Q) at the P1 position, along with a bulky hydrophobic residue at P2. Given their predominant endosomal/lysosomal location there is a distinct requirement of low pH for enzymatic activity. Cathepsin L (Fig. 2B) is most commonly associated with activation of viral glycoproteins, and has a pH optimum of 3.0 to 6.5. Cathepsin L is known to process SARS-CoV S, as well as a range of other coronaviruses, including MERS-CoV, HCoV-229E, and MHV-2 (Kawase et al., 2009, Qiu et al., 2006, Shirato et al., 2013, Simmons et al., 2005). Cathepsin B can also be involved in coronavirus entry, and has some distinct properties compared to cathepsin L and other cathepsins; it has a generally higher pH optimum and, while preferring an aromatic P2 residue, can process di-basic substrates, in contrast to the other cathepsins (Choe et al., 2006, Mort, 2004, Polgár, 1989). Coronaviruses using cathepsin B for entry include type II feline coronavirus (FCoV) and MHV-2 (Qiu et al., 2006, Regan et al., 2008).

3.2.4. Other proteases: Transmembrane protease/serine (TMPRSS) proteases, elastase, plasmin

Members of the TMPRSS family are membrane-bound tryspin-like serine proteases that are found on the cell surface or in the secretory pathway of cells (Antalis et al., 2011, Simmons et al., 2013). TMPRSS proteases are part of the larger family of proteases, the type II transmembrane serine proteases or TTSP (Bugge et al., 2009). TTSPs are subdivided into four groups: the HAT/DESC, Hepsin/TMPRSS, Matriptase, and Corin subfamilies. TMPRSS proteases are type II transmembrane proteins. They are widely expressed in the respiratory tract and are involved in the activation of many respiratory viruses. Both MERS-CoV S and SARS-CoV S can be activated by TMPRSS family members, as well as HCoV-229E S (Bertram et al., 2013, Gierer et al., 2013, Glowacka et al., 2011, Shirato et al., 2013). Intriguingly, TMPRSS2 was shown to be important for the late stages of PEDV life cycle (Shirato et al., 2011). While the substrate specificity of TMPRSS proteases is not well defined, it is generally thought to be similar to that of trypsin.

Recently, the TTSPs mosaic serine protease large form (MSPL), and differentially expressed in squamous cell carcinoma 1 (DESC1) were shown to activate MERS-CoV and SARS-CoV S proteins for cell–cell and virus–cell fusion (Zmora et al., 2014). MSPL and DESC1 were also shown to activate the HA proteins of influenza virus. The authors demonstrated by quantitative RT-PCR that while MSPL was readily expressed in human lung tissue, DESC1 was only expressed at low levels. Remarkably, TMPRSS13, a splice variant form of MSPL, was shown to be sensitive to inhibition by the naturally occurring Kunitz-type serine protease inhibitor, hepatocyte growth factor activator inhibitor type 1 (HAI-1) (Hashimoto et al., 2010). HAI-1 was shown earlier to be involved in the regulation and inhibition of TTSPs such as hepsin, matriptase and prostatin (Fan et al., 2005, Kirchhofer et al., 2005, Lin et al., 1999). A related protease inhibitor, HAI-2 was shown to inhibit influenza virus infection in vitro and its administration protected animals in a mouse model for influenza infection (Hamilton et al., 2014). It would be of interest to test such naturally occurring inhibitors for their effects on coronavirus entry and infection.

Elastase has been shown to activate SARS-CoV S. While the biological relevance of this has focused on elastase produced by neutrophils during the inflammatory response, most experimental studies have used pancreatic elastase. Like trypsin, pancreatic elastase can shift SARS-CoV entry to a low pH-independent route (Belouzard et al., 2010, Matsuyama et al., 2005), although the amino acid preferences for elastase are very different to trypsin, with alanine (A), serine (S), glycine (G) and valine (V) residues being preferred at the P1 position (Bieth, 2004).

Plasmin is a key enzyme in blood clot lysis and its major natural substrates are fibrinogen and fibrin (Castellino, 2004, Castellino and Ploplis, 2003). It is produced from its precursor plasminogen. Plasmin is also involved in a variety of other cellular processes in various tissues. Like trypsin, plasmin cleaves at K- and R-bonds, but is much less efficient and cleaves only a subset of such bonds. In part, this is due to differences in the P2 residues, where bulky hydrophobic residues are preferred (Backes et al., 2000, Hervio et al., 2000). Thus plasmin is a much more selective enzyme than trypsin. Plasmin is known to activate certain influenza virus HAs (Lazarowitz et al., 1973b, Sun et al., 2010, Tse et al., 2013). Although plasmin has been shown to activate SARS-CoV S (Kam et al., 2009), there remains little direct biological evidence for a role of plasmin in activating SARS-CoV or other coronaviruses.

Another possible source of proteases that may impact coronavirus pathogenesis is proteases produced by co-infecting bacteria. Many bacterial species secrete proteases as virulence factors, or elicit secretion of host proteases, and these proteases are likely to have the capacity to activate virus envelope glycoproteins under certain co-infection conditions. Such a situation has been explored for influenza virus (Böttcher-Friebertshauser et al., 2013), but has not generally been evaluated in coronaviruses, except for infection of mice with SARS-CoV and Pasteurella pneumotropica, a low-pathogenicity bacterium that elicits elastase production in the lungs (Ami et al., 2008).

3.4.1. Severe acute respiratory syndrome coronavirus (SARS-CoV)

The emergence of SARS-CoV in the human population reinforced research in coronavirus biology. In particular, the entry pathways and activation of SARS-CoV, a betacoronavirus, have become the subject of intense study. The proteolytic activation mechanisms of SARS-CoV S protein are perhaps amongst the best characterized so far and provide a good model to investigate coronavirus S proteolytic processing because many details of the proteases involved and cleavage sites processed are known. SARS-CoV also represents an example of how virus envelope glycoproteins can be activated by a diverse array of mechanisms.

Initially, a study employing retroviral pseudovirions produced in HEK-293T cells and harboring SARS-CoV S has found that SARS-CoV entry was sensitive to lysosomotropic agents, indicating a role for endosomal low pH (Simmons et al., 2004). Using similar HEK-293T produced SARS-CoV S pseudovirions, it was later shown that the dependency of virus entry on low-pH was because SARS-CoV S can be activated by endosomal cathepsin L, which requires low pH for its enzymatic activity (Simmons et al., 2005). Surprisingly, SARS-CoV seems to have evolved a multi-pronged approach regarding the activation of its S as many other proteases were shown to enable S-mediated entry and fusion, most of which act on the virus outside of the cell. Cathepsin L was shown to cleave SARS-CoV S at residue T678 in the S1/S2 boundary (Table 1) (Bosch et al., 2008). Trypsin was shown to activate S-induced syncytia formation, without the need to acidify the extracellular milieu (Simmons et al., 2004), indicating that the low pH requirement observed for SARS-CoV entry is not due to S requiring low pH for fusion competency. Trypsin was found to cleave SARS-CoV S at R667 in the S1/S2 site (Table 1) (Li et al., 2006). Furthermore, using native SARS-CoV infection in VeroE6 cells, it was shown that along with trypsin, thermolysin and elastase were also able to activate SARS-CoV fusion and entry (Matsuyama et al., 2005). The authors showed that cleavage activation by trypsin and thermolysin on virions bound on the cell surface allowed a much more efficient entry (>100-fold enhancement of infectivity) than endosomal cathepsin-mediated entry. Notably, prior treatment of virions before cell attachment decreased infectivity. This result suggests that fusion-active cleaved SARS-CoV S proteins are unstable, and the conformational changes induced by the cleavage event are transient and irreversible.

The location of the cleavage sites within SARS-CoV S has been investigated. Using wild type and mutated SARS-CoV S pseudovirions produced in HEK-293T cells, it was found that S could be cleaved by trypsin at two distinct sites, one located at the boundary of S1 and S2, the “classical” S1/S2 site (R667 P1 residue), and another newly identified site, S2′ (R797 P1 residue), found upstream of the putative fusion peptide and within the S2 domain (Table 1, Table 2) allowing for activation of fusion and entry (Belouzard et al., 2009). Importantly, trypsin cleavage of SARS-CoV S is thought to be sequential, with the S1/S2 cleavage occurring first and enhancing subsequent cleavage at S2′. It is the second cleavage event, at S2′, that is believed to be crucial for fusion activation of S. The S1/S2 cleavage appears dispensable for syncytia formation and virus–cell fusion.

Elastase-mediated cleavage was shown to occur at the S2′ site of SARS-CoV S on pseudovirions produced in HEK-293T cells, with the P1 residue found to be a threonine, T795 (Table 2) (Belouzard et al., 2010). Cleavage at this site shifts the starting position of the putative fusion peptide, which modulates but does not abrogate fusogenicity of S. A study has shown that plasmin, a known protease activator of influenza HA, could, along with trypsin, cleave SARS-CoV S expressed in BHK-21 cells (Kam et al., 2009).

The respiratory-tract resident and cell surface-expressed TMPRSS2 and TMPRSS11a proteases were shown to cleave and mediate activation of SARS-CoV entry in studies using HEK-293T-produced SARS-CoV S pseudoviruses (Glowacka et al., 2011, Kam et al., 2009). This is a feature shared with influenza HA, which is known to be activated by TMPRSS proteases (Böttcher et al., 2006). Remarkably, it was found that TMPRSS2 associates with the SARS-CoV receptor, ACE2, forming a receptor-protease complex allowing for efficient entry of the virus, directly at the cell surface (Shulla et al., 2011). Another TMPRSS protease, TMPRSS11d also known as human airway trypsin-like protease (HAT), was also shown to proteolytically activate SARS-CoV S in a HEK-293T expression system (Bertram et al., 2011). Using mass spectrometry, the authors demonstrated that TMPRSS11d cleaved S at R667, in the S1/S2 cleavage site (Table 1). While TMPRSS2 cleavage appears to occur at different sites within S, TMPRSS11d cleavage occurs mostly at the S1/S2 site (R667), which may explain why TMPRSS11d activation appears sufficient for cell–cell fusion but not virus–cell fusion. After TMPRSS2 cleavage, shed fragments of S, including a large 150 kDa fragment, are detected in the supernatant of cells and were shown to act as decoys to anti-S neutralizing antibodies (Glowacka et al., 2011).

Factor Xa is yet another protease that was shown to activate SARS-CoV S for entry into host cells (Du et al., 2007). The authors showed that the S protein of SARS-CoV retroviral pseudovirions produced in HEK-293T cells could be cleaved by factor Xa at the S1/S2 boundary. This cleavage event occurred when the pseudovirions were incubated with susceptible cells that were shown to express the protease.

Overall, the study of SARS-CoV S was instrumental in defining cleavage sites and putting forward the notion that the coronavirus S protein can be cleaved by a wide variety of proteases, suggestive of a relatively high degree of plasticity in cleavage mechanisms employed.

3.4.2. Murine hepatitis virus (MHV)

The betacoronavirus MHV has been extensively studied and represents a good model for coronavirus biology and in particular for studying coronavirus entry mechanisms. In groundbreaking work on MHV strain A59 (MHV-A59), the S protein found on virions (then known as E2), was shown to be cleaved during its biosynthesis when produced in four infected murine cell lines: 17 Cl 1, Sac^-, DBT and L2 (Frana et al., 1985). Pulse-chase experiments demonstrated that the cleavage event occurred before virion release from producer cells. The authors showed that the degree of cleavage varied depending on which cell type the S protein was expressed in. A study from the same group showed that the 180 kDa precursor S protein of MHV-A59 virions produced in the 17 Cl 1 mouse cell line was cleaved approximately in two halves, with equal amounts of uncleaved and cleaved S and yielding two ∼90 kDa cleavage products (Sturman et al., 1985). The proteolytic processing of S was found to be potentiated by trypsin treatment of virions produced in murine 17 Cl 1 cells. The site where trypsin cleaved MHV-A59 S was mapped to the S1/S2 site (Table 1) (Luytjes et al., 1987). It was later shown that MHV-A59 S is cleaved by furin during biosynthesis and trafficking of S through the trans-Golgi network in murine LR-7 cells (de Haan et al., 2004). MHV-A59 S contains a minimal furin cleavage motif (R-R-A-H-R-S), containing at least P1 and P4 arginine residues (R-X-X-R) at the S1/S2 boundary, a feature that is shared by some alphacoronaviruses and many other coronaviruses from the beta- and gamma-genera (Table 1). Mutating the S1/S2 site was shown to greatly affect the degree of cleavage of the S protein (de Haan et al., 2004). Another important concept that comes out from MHV studies is that the degree of cleavage at S1/S2 found when S is expressed and matures in different cell lines reflects the varying abundance of protease expression.

The MHV-2 strain was found to have lost this cleavage site and its S is uncleaved on virions exiting murine DBT producer cells (Yamada et al., 1997). When expressed in cells, MHV-2 S fails to induce syncytia at neutral pH. MHV-2 S-induced syncytia can form if exogenous protease treatment is applied. It appears that MHV-2 still requires proteolytic processing for fusion competency. Indeed, MHV-2 S-mediated entry of virions produced in 17 Cl 1 murine cells is sensitive to inhibitors of the endosomal compartment resident proteases cathepsin B or L in murine fibroblast L2 target cells (Qiu et al., 2006). This suggests that for MHV-2, the timing and nature of proteases involved has been switched from furin-like proteolytic processing during virus maturation in producer cells to endosomal cathepsins during entry in target cells.

Recent work reported that the S protein of MHV-A59, in addition to being cleaved at S1/S2 during protein biosynthesis in mouse LR7 producer cells, is cleaved a second time at a site near the putative fusion peptide, named S2*, during the viral entry process (Wicht et al., 2014a). This site is analogous to the S2′ site in SARS-CoV S (Belouzard et al., 2009). The authors devised a clever experimental setup using conditional biontinylation assay that allows specific labeling and detection of MHV S proteins that have undergone the fusion process. This allows the analysis of any cleavage activation that may have occurred before fusion takes place. A new ∼80 kDa band was detected by Western blot analysis, which the authors conclude to be the fusion subunit. A comprehensive study, using siRNA screenings and a replication-independent fusion assay, has shown that MHV-A59 entry necessitates clathrin-mediated endocytosis and that the virions fuse with lysosomal membranes (Burkard et al., 2014). The study also conclusively demonstrates that MHV-A59 requires cleavage of S, at the S2′ site. Inhibition of MHV-A59 entry could be obtained by using a pan-lysosomal protease inhibitor. The identity of the specific protease involved awaits elucidation.

3.4.3. Bovine coronavirus (BCoV)

A recent study on the betacoronavirus BCoV has uncovered important features of coronavirus population adaptation during host, tissue, and cell tropism shifts (Borucki et al., 2013). Intriguingly, the authors found a critical role for a genotype containing a S variation at the S2′ site that rapidly expands and dominates the population during the course of passaging in cell culture (Table 3 ). Using a deep-sequencing approach the authors looked at the effect of passaging field strains of BCoV obtained in nasal washes on the genotype composition of the population. The viruses were passaged in human or bovine macrophage cell lines (THP-1 and BOMAC) as well as in a human rectal cell line (HRT-18). Surprisingly, they found that a variant genotype, a minority present in unpassaged samples, and which harbored a mutated S containing a multibasic insert composed of three arginines at S2′, was independently selected during passaging of three different nasal wash samples in either BOMAC, THP-1 or HRT-18 cell lines (for example, the B2.27.BO.P1 BOMAC passaged virus, Table 3). Surprisingly, the study showed that just a single passage in BOMAC cells was sufficient to expand the minority variant to become dominant in the viral population. It is notable that the insertion of the three basic arginines (R-R-R) is very similar to what is observed in the HA polybasic sites of highly pathogenic avian influenza virus subtypes H5 and H7 (Kawaoka and Webster, 1988, Klenk and Garten, 1994a, Lee et al., 2006). The work on BCoV demonstrates the important role of cleavage sites in shifting coronavirus genetic populations depending on the proteolytic environment.

3.4.4. Infectious bronchitis virus (IBV)

The avian gammacoronavirus IBV was the first described coronavirus. It provokes extensive impairment of respiratory and urogenital tracts of chickens, and has been well studied. Many strains of IBV have been characterized, most of which have a restricted cell and tissue tropism, often limited to primary chicken cells. All IBV strains harbor a well-defined furin cleavage site at the S1/S2 site (R-R-F-R-R-S, Table 1) that is cleaved during protein biosynthesis. Strikingly, a lab adapted strain of IBV, the Beaudette strain, which has been heavily egg- and cell-culture adapted has acquired a mutation at its S2′ site, giving rise to the following motif S-R-R-R/K-R-S. Such a site is deemed ideal for furin recognition and cleavage as it harbors the critical P1 and P4 arginines (R), along with a highly favorable basic P2 lysine (K) or arginine (R) as well as flanking serines (S) at P1′ and P6 (Yamada and Liu, 2009). Notably, the IBV-Beaudette S2′ site is also cleaved during protein biosynthesis in S-transfected Huh-7 cells and in IBV-Beaudette-infected Vero cells, and this has been shown to be important for syncytia formation as well as entry of the virus (Belouzard et al., 2009, Yamada and Liu, 2009). Furthermore, IBV-Beaudette has a characteristically widened tissue and cell tropism and can productively infect many different cell types in vitro, including human cells. Importantly, high levels of furin expression, such as that observed in Huh-7 cells, was found to correlate tightly with highly productive infection by IBV-Beaudette produced in Vero cells (Tay et al., 2012). The wide cell and tissue tropism is in sharp contrast with the very restricted cell and tissue tropism observed for the other strains of IBV, which do not harbor a S2′ furin cleavage site. Similarly to the concept found for avian influenza, modification of its S2′ cleavage site may have allowed IBV-Beaudette to be cleaved by ubiquitously expressed furin, allowing for this expansion in tissue and cell tropism. This unusual feature is shared with MERS-CoV, as described in paragraph 2.4.7. Middle East respiratory syndrome coronavirus (MERS-CoV).

3.4.5. Porcine epidemic diarrhea virus (PEDV)

An alphacoronavirus, PEDV causes severe outbreaks of diarrheal disease in pigs mostly centering in East Asia (Park et al., 2011b). The virus recently emerged in the United States and is of particular concern because of the high mortality rates associated with piglet infections. In an early study, trypsin was found to activate PEDV for infection in Vero cells (Hofmann and Wyler, 1988). The S protein of Vero cell-produced PEDV was shown to be cleaved by trypsin when the virus is engaged with its receptor, but not on free virions (Park et al., 2011a). This suggests that PEDV S protein requires receptor-binding-induced conformational changes in order for trypsin cleavage to occur. TMPRSS2 was found to play a role in releasing PEDV particles from Vero producer cells stably expressing TMPRSS2 (Shirato et al., 2011). PEDV produced in Vero cells was also shown to enter cells via a clathrin-dependent endocytic pathway, also involving low pH and the activity of serine proteases (Park et al., 2014). In a recent study, notable findings about proteolytic activation of PEDV S have been uncovered (Wicht et al., 2014b). The authors focused on analyzing and comparing two strains of PEDV propagated in Vero cells, the first, CV777 which is an isolate that, like most PEDV isolates, requires trypsin activation for infection in cell culture, and the other, DR13, is a cell culture adapted strain, and has evolved to become trypsin-independent. The authors were able to demonstrate that CV777 S harbored a critical arginine (R) residue at the S2′ site that is cleaved by trypsin (Table 2). Notably, they found that in the cell-culture adapted strain DR13, an R to G mutation occurred at the S2′ site and explains why the strain has evolved to become trypsin-independent. As most coronavirus S proteins contain an arginine (R) at the S2′ site (Table 2), it would be interesting to study whether the findings of Wicht et al. can be generalized.

3.4.6. Feline coronavirus (FCoV)

Studies on FCoV offer deep insights into the role of S and in particular its proteolytic processing and fusion activation in coronavirus pathogenesis and host cell tropism. The FCoV is an alphacoronavirus and there are two known serotypes, based on differential S antigenicity (Pedersen, 2009). While serotype II viruses are the most extensively studied, notably due to their propensity to grow well in vitro, such as in CrFK feline kidney cells, they are not the most clinically prevalent. The serotype I is the most prevalent but is difficult to culture and so has yet to be extensively studied, although some strains, such as Black (TN-406) and UCD1, can be grown in feline AK-D cells and feline macrophage-like cell line Fcwf-4, respectively (Neuman et al., 2006, Pedersen, 2009). In both serotypes I and II, FCoV exists as two biotypes or pathotypes that display extremely contrasting pathogenicity. The low pathogenicity biotype, feline enteric coronavirus (FECV) is a highly prevalent virus that transmits easily through the fecal-oral route and generally causes only mild and self-limiting enteritis. In contrast, the highly pathogenic biotype, feline infectious peritonitis or FIPV, causes an acute, systemic and immune-mediated disease that is invariably fatal and represents a leading cause of infectious disease deaths in cats. The current understanding on FIP pathogenesis, the so-called “internal mutation theory”, is that it arises from an initial FECV infection in which the virus acquires mutations within the infected animal that leads to its conversion from the low pathogenic form to the virulent FIPV form (Vennema et al., 1998). A key difference between FECV and FIPV is their tissue and cell tropism: while FECV is generally restricted to the epithelial cells of the gastrointestinal tract, FIPV has the ability to readily infect monocytes and macrophages (Stoddart and Scott, 1989). It is this change in cell and tissue tropism, from gut epithelial cells to motile immune cells, that is thought to be the crucial step toward systemic spread of the virus. Several studies have mapped mutations that are linked to the transition between the biotypes, located in the accessory 3c and ORF 7 genes (Herrewegh et al., 1995, Pedersen et al., 2012), but there is a growing body of evidence indicating that mutations within the S protein are key to the development of FIP (Chang et al., 2012, Licitra et al., 2013). In the following section we will describe the current understanding on the S mutations that are thought to be linked with FIP.

3.4.7. Middle East respiratory syndrome coronavirus (MERS-CoV)

MERS-CoV, a recently emerged virus from the Middle East that is associated with severe pneumonia with a high case fatality rate, is another example of a coronavirus whose S protein can be activated by a multitude of proteases. Since its isolation and discovery in 2012, the complete viral genome was swiftly sequenced (van Boheemen et al., 2012), and its receptor, dipeptidyl peptidase 4 (DPP4 or CD26), rapidly identified (Raj et al., 2013).

In a report using pseudovirions produced in HEK-293T cells as well as protease inhibitors, it was first shown that TMPRSS2 and endosomal cathepsins could activate MERS-CoV virus–cell fusion (Gierer et al., 2013). The authors also reported the sensitivity of MERS-CoV entry to the lysosomotropic weak base NH₄Cl, a result attributed to a decreasing of activity of endosomal cathepsins, which require low pH.

The role of cell surface-expressed TMPRSS2 in the activation of MERS-CoV was confirmed using native MERS-CoV virions produced in Vero cells, and it was estimated that the entry of the virus in Vero cells overexpressing the protease was increased 100-fold, compared to control cells (Shirato et al., 2013). The authors described how TMPRSS2 could greatly potentiate MERS-CoV infection-induced cell–cell fusion in Vero cells stably expressing the protease. The study also corroborated that endosomal cathepsin L could activate MERS-CoV S. Notably, the study showed that depending on the target cell type, MERS-CoV could use different activation pathways.

Along with TMPRSS2, TMPRSS4 was also shown to activate MERS-CoV S-mediated cell–cell fusion in a co-transfection HEK-293T cell expression system (Qian et al., 2013).

More recently, a study has revealed that MERS-CoV S from native virions produced in Vero cells could be cleaved by furin at two distinct sites, S1/S2 and S2′, with the former cleavage occurring during S biosynthesis in the Vero producer cells, and the latter, during virus entry into target cells (Millet and Whittaker, 2014). Similarly to the situation with IBV-Beaudette, it was also shown that expression of high levels of furin, such as in Huh-7 cells, along with the receptor DPP4, is associated with increased susceptibility to MERS-CoV infection. The role of furin during entry of MERS-CoV was confirmed by a recent study by Burkard and colleagues (Burkard et al., 2014). Intriguingly, a similar two-step activation mechanism for entry by furin was demonstrated for the paramyxovirus respiratory syncytial virus (RSV) (Gonzalez-Reyes et al., 2001, Krzyzaniak et al., 2013, Zimmer et al., 2001). It was shown for bovine RSV that the dual cleavage events generated a small peptide fragment, the virokinin, which is released upon activation and entry and has bioactive properties implicated in exacerbating pathogenesis (Valarcher et al., 2006, Zimmer et al., 2003).