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Review Intervirology Three Main Inducers of Alphacoronavirus Infection of Enterocytes Sialic Acid Proteases and Low pH Peng Yuan Zhou Yang Han Song Kai Wang Yang Yang Luyi Xie Shilei Huang Jia Liu Lin Ran Zhenhui Song Department of Veterinary Medicine College of Animal Science Southwest University Chongqing PR China Received November 29 2017 Accepted July 19 2018 Published online September 3 2018 Zhenhui Song PhD Department of Veterinary Medicine College of Animal Science Southwest University Chongqing 402460 People s Republic of China E Mail szh7678 2018 S Karger AG Basel E Mail karger DOI 10 1159 000492424 Keywords Sialic acid Protease Low pH Transmissible gastroenteritis virus Porcine epidemic diarrhea virus Porcine small intestine Epithelial cells Abstract Transmissible gastroenteritis virus TGEV and porcine epi demic diarrhea virus PEDV are similar coronaviruses caus ing diseases characterized by vomiting diarrhea and death from severe dehydration in piglets Thus they have caused huge losses to the swine breeding industry worldwide Nowadays they are easily transmitted among the conti nents via vehicles equipment and cargo Both viruses estab lish an infection in porcine enterocytes in the small intestine and their spike S proteins play a key role in the virus cell binding process under unfavorable conditions when the in testine with a low pH is filled with a thick layer of mucus and proteases Sialic acid proteases and low pH are three main inducers of coronavirus infection However the details of how sialic acid and low pH affect virus binding to the host cell are not determined and the functions of the proteases are unknown This review emphasizes the role of three fac tors in the invasion of TGEV and PEDV into porcine entero cytes and offers more insights into Alphacoronavirus infec tion in the intestinal environment 2018 S Karger AG Basel Introduction Two porcine coronaviruses transmissible gastroen teritis virus TGEV and porcine epidemic diarrhea virus PEDV are clustered as different species into the Al phacoronavirus genus They both are important viral pathogens in piglets causing similar pathological charac teristics with acute diarrhea and dehydration 1 2 lead ing to massive losses in the modern swine breeding in dustry worldwide Although their transmission route is limited to the fecal oral route as economic globalization increases rapidly and transportation develops remark ably vehicles equipment and cargo have been conve nient for these viruses to spread to all continents TGEV and PEDV replicate in enterocytes of the small intestine and are the causative agent of a fatal diarrhea in newborn piglets Doyle and Hutchings 3 described the Yuan Yang Song Wang Yang Xie Huang Liu Ran Song Intervirology 2 DOI 10 1159 000492424 disease caused by TGEV in America for the first time TGEV was then spreading to various continents North America Canada 1989 Europe England 1957 and Asia Japan 1956 Korea 2000 and Thailand 2000 In China TGEV was reported in the 1960s and widespread outbreaks have occurred since 2010 4 In the 1970s PEDV was first reported in Europe 5 6 Subsequently the virus affected Asian countries extensively and PEDV infection was usually mild before 2010 The current viru lent PEDV strains appeared in 2010 in China Then an outbreak occurred in Ohio in the USA in 2013 which spread throughout the USA Since then PEDV has re sulted in significant economic damage worldwide and is thus receiving increased attention around the world 7 TGEV and PEDV cluster in the genus Alphacorona virus which belongs to the Coronavirinae subfamily Fig 1 order Nidovirales family Coronaviridae Coro naviruses CoVs are enveloped viruses with a single stranded positive sense RNA genome of up to approxi mately 30 kb with a 5 cap and a 3 polyadenylated tail 8 They are capable of cross species transmission and may gain new features CoVs are significant infectious agents involved in gastroenteric respiratory hepatic and neu ronal diseases in animals and humans causing cough and diarrhea as well as high mortality rates and bringing huge damage to human health and society 9 and CoVs infect mammals such as humans and swine By contrast and CoVs are mostly detected in birds such as Bulbul coronavirus HKU11 Thrush coronavirus HKU12 and Munia coronavirus HKU13 Both and CoVs have also been found in mammals 10 and CoVs are 4 genera of Coronaviridae that are clustered together based on numerous studies and sero logical and genotypic criteria Table 1 Each genus has its own representative CoV species TGEV PEDV and human coronaviruses HCoV 229E and HCoV NL63 are typical viruses of the genus CoV The representa tive species of CoV are mouse hepatitis virus and se vere acute respiratory syndrome coronavirus SARS CoV Infectious bronchitis virus is currently the most studied virus in the CoV genus 11 Little is known about CoV This review emphasizes the role of three factors sialic acid proteases and low pH in the invasion of TGEV and PEDV into porcine small intestine epithelial cells and provides information with respect to CoV infection that brings new insights into virus research The Role of the Spike Protein of TGEV and PEDV The spike S protein of CoVs is essential for the inter action with receptors and the fusion of the viral particles and for cellular membranes It also plays a crucial role in the interspecies transmission of CoVs The interactions of CoV S glycoproteins with receptors on the cell surface determine the host range and tissue tropism of CoVs 12 13 Virus infection begins with the interplay between the Table 1 Representative prototypes of coronaviruses with their own receptor or coreceptor Genus Representative prototypes Receptor coreceptor Alphacoronavirus TGEV pAPN Neu5Ac Neu5Gc HCoV 229E hAPN HCoV NL63 ACE2 PEDV pAPN Neu5Ac Betacoronavirus MERS CoV DPP4 SARS CoV ACE2 BCoV Neu5 9Ac2 MHV CEACAM1 HCoV OC43 Neu5 9Ac2 Gammacoronavirus IBV Neu5Ac Deltacoronavirus Bulbul coronavirus HKU11 Unidentified Thrush coronavirus HKU12 Munia coronavirus HKU13 In the table each color line corresponding to one species is listed in the order as presented in the phylogenetic tree The full names of the species in the table have been mentioned except for bovine coronavirus BCoV and human coronavirus OC43 HCoV OC43 The full names of the protein receptors and sugar receptors are shown in the table Color version available online Inducers of Alphacoronavirus Infection of Enterocytes 3 Intervirology DOI 10 1159 000492424 Tree scale 1 Fig 1 Phylogenetic tree of the Coronavirinae subfamily The phy logenetic tree was built on the basis of the nucleotide sequences of complete spike genes from 213 coronaviruses The nucleotide se quence alignment and the construction of the phylogenetic tree were completed using the MEGA5 0 program with a proper sub stitution model d transitions transversions and all other set tings were maintained as default The final depiction of the phylo genetic tree was completed using iTOL on the Internet As the map shows the coronavirus family is divided into 4 main groups and 11 coronaviruses are regarded as representatives which are shown in various colors Interestingly the spike gene of PEDV isolate strain ZJ14HZ030301 is separated into a single group in the phy logenetic tree Color version available online Yuan Yang Song Wang Yang Xie Huang Liu Ran Song Intervirology 4 DOI 10 1159 000492424 S protein and its specific receptors followed by penetra tion into the cells by a fusion event 14 15 The S protein a class I fusion protein is a membrane protein It is the largest glycoprotein of CoVs 16 pro jecting out from the surface of CoV particles and forming a homotrimer structure called a peplomer The S protein is responsible for the corona like appearance of the sur face projections in the electron microscope The peplomer includes a globular portion and a protein stalk By adopt ing the helical structure that is characteristic of class I vi rus fusion proteins the protein stalk connects the globu lar portion to the transmembrane domain 17 The N terminal S1 domain constitutes the globular region and the stalk is made up of the membrane proximal S2 do main The N terminal S1 domain and C terminal S2 do main of the S protein play a similar role in all CoVs the S1 region is related to receptor binding and the S2 do main plays a role in the membrane fusion process In ad dition the S1 domain contains two subdomains an N terminal domain NTD and a C terminal domain CTD Fig 2 The two subdomains called RBDs receptor binding domains bind with specific cell receptors in cluding a series of proteins and sugars Thus determi nants in the S1 domain are not only crucial for initiating virus entry into cells they also determine the cell and host tropism of CoVs 12 TGEV and PEDV S proteins have many similarities in their secondary structure The TGEV S protein is pro duced as a 1 447 amino acid precursor polypeptide with a 16 residue signal peptide According to analysis of the S protein sequences of CoVs with different biological phenotypes there are 4 antigenic sites C B D and A in that order in the N terminal half of the S protein among which the A site can induce neutralizing antibodies 4 and is highly conserved in TGEV and porcine respira tory coronavirus PRCoV a respiratory variant of TGEV The S protein encoded by PRCoV lacks about 200 amino acids in the N terminal region that contain determinants related to the enteropathogenicity of TGEV 18 PEDV has a 150 to 220 kDa spike glyco protein with a homotrimeric structure The PEDV S protein is also a type I transmembrane glycoprotein that contains 5 regions a signal peptide residues 1 20 an S1 region residues 21 793 an S2 region residues 794 1385 a transmembrane domain residues 1335 1358 and a cytoplasmic tail residues 1359 1385 19 The S1 region is responsible for virus particle binding to the cel lular receptors whereas the S2 region participates in membrane fusion of the virus and host cells Like other CoVs the S1 region possesses two subdomains compris ing an NTD residues 21 324 and a CTD residues 253 638 The CTD of the S1 domain binds to a func tional cellular receptor for PEDV infection Sequence analyses of PEDV prototype and variant strains reveal that the N terminus of the S protein changes more eas ily than the C terminus In addition a previous report suggested an interaction of NTD of the S1 domain with a coreceptor 20 21 Fig 2 Simplified graphic of the structural domains of the main coronaviruses spike proteins The spike protein structure can be divided into the S1 and S2 domains and the structural domains in the spike protein are located in the order from C to the N terminus as transmembrane TM hep tad repeats HRs in the S2 domain C ter minal domain CTD and N terminal do main NTD in the S1 domain as well as the signal peptide SP In the graphic com pared with TGEV PRCoV lacks an NTD which is associated with enteric tropism The RBD in the SARS spike protein is shown as discontinuous in the graphic Color version available online Inducers of Alphacoronavirus Infection of Enterocytes 5 Intervirology DOI 10 1159 000492424 The Conformation of the RBD in TGEV and PEDV The CoV RBD comprises about 150 residues that ad join the CTD in the S1 region Fig 2 Previous studies on the CTD concluded that the structure had the characteris tic of independent expression of the S protein and preser vation of its native structure maintained the binding spec ificity 4 Several structural studies indicate that the CoV RBD adopts a barrel fold with 2 highly twisted sheets in which 3 strands 1 3 and 7 run parallel and 3 di sulfide bonds exist in the 3 structures 22 The RBD of TGEV is located within the CTD of the S1 domain 23 In the TGEV RBD crystal structure the bent strand 5 cross es both sheets N linked glycans are concentrated at one side of the barrel the opposite side is not glycosylated and might be closer to other S protein domains The N and C terminal ends of the RBD are located on the same side of the domain terminal side at the opposite side 2 turns form the tip of the barrel in the TGEV RBD 12 Like TGEV the crystal structure of a single domain unit in the PRCoV RBD adopts a barrel fold with 2 high ly twisted sheets located in the CTD of the S1 domain and engages in binding to the host cell surface receptor Compared with the TGEV S domain the related PRCoV lacks the NTD which is related to enteric tropism The TGEV or PRCoV RBD tips consist of 2 protruding turns 1 2 and 3 4 each having a solvent exposed aro matic residue tyrosine or tryptophan 20 In the tertiary structure of the PRCoV RBD 3 loops 1 2 3 4 and 5 6 at the tips of the barrel domains are responsible for receptor binding Some researchers found that single amino acid mutations in the 3 loops completely or sig nificantly reduced the ability of PRCoV RBD to bind to host receptors and mutations outside the RBD had no ef fect on receptor recognition 24 To date there have been few reports on the RBD struc ture of PEDV It is interesting that 3 receptor binding mutant proteins RBM1 1 RBM2 1 and RBM3 2 pro teins did not significantly reduce PEDV pAPN binding activities in virus infection 4 suggesting the PEDV S protein uses a receptor binding mechanism different from TGEV and PRCoV PEDV is further confirmed to have a broader receptor range than other CoVs 21 Cell Entry Receptors of TGEV and PEDV During the progress of evolution and adaptation to di verse hosts CoVs have evolved to use various receptors to enter host cells Different hosts or virus strains produce evolutionary diversity in the same virus family and the binding of CoVs to susceptible cells seems to show varia tion in the receptors used that correspond to viral groups and species These CoVs recognize distinct cellular recep tors and coreceptors such as proteins and sugars to fa cilitate their penetration into cells 8 Currently there are 4 main protein receptors APN angiotensin convert ing enzyme 2 ACE2 carcinoembryonic antigen related cell adhesion molecule 1 CEACAM1 and dipeptidyl peptidase 4 DPP4 Most members of CoV use APN as the receptor for infecting host cells such as TGEV PEDV and HCoV 229E APN also known as CD13 is a 150 kDa type II transmembrane protein that belongs to a mem brane bound metalloprotease family 24 Interestingly HCoV NL63 as an CoV shares the same cell entry re ceptor identified as ACE2 with SARS CoV which is a CoV ACE2 is a type I integral membrane glycoprotein with an N terminal extracellular domain comprising 2 helical lobes between both of which there is a catalytic site with a coordinated zinc ion 25 By contrast the CoV mouse hepatitis virus utilizes CEACAM1 as a cell surface receptor for the S protein CEACAM1 the first identified CoV receptor 26 is a type I transmembrane multifunctional protein and a member of the immuno globulin superfamily termed IgSF Middle East respira tory syndrome related CoV belonging to CoV has been shown to use DPP4 as a cell entry receptor DPP4 also known as CD26 a type II membrane protein is a multifunctional membrane bound serine protease that forms homodimers on the surface of host cells The DPP4 ectodomain comprises about 730 amino acids and has 2 domains an hydrolase domain and an 8 bladed pro peller 27 Among these cellular receptors and corecep tors APN is a major cell entry receptor for CoVs APN exists on the epithelial cell surface of different tissues In particular it is expressed abundantly in the brush border membrane of the small intestine the kidney and the re spiratory tract 28 Most CoVs use APN as cell entry receptor For example previous studies showed that TGEV uses porcine p APN as the receptor in the entry process 24 whereas human h APN is a receptor for the infection by HCoV 229E 29 The reason why TGEV uses APN as an entry receptor has not been clarified It might be linked to its abundance on the surface of epithelial cells rather than its biological function which seems to be dispensable for CoV binding capacity 30 31 In the small intestine mucosa APN oc cupies about 8 of the total protein content of the differ entiated enterocytes 32 For PEDV although there are many articles in which pAPN was proposed as the recep Yuan Yang Song Wang Yang Xie Huang Liu Ran Song Intervirology 6 DOI 10 1159 000492424 tor in PEDV infection this view has been questioned due to the lack of robust direct evidence The characteristic structure of APN is a large glycosylated ectodomain with a zinc metal ion at the active site which functions as a zinc dependent protease responsible for cleavage of the N terminal amino acids mediated by the HELAH motif 33 The enzymatic function of the pAPN catalyzes the removal of amino acid residues from the N termini of oli gopeptides and APN has been termed the moonlighting enzyme because of its many cell functions APN can be cleaved into N terminal 95 kDa and C terminal 50 kDa subunits by trypsin digestion and comprises 4 do mains DI DIV 29 32 It is heavily glycosylated and forms dimers through extensive DIV DIV interactions Sequence conservation in the RBD tip of CoVs exerts a crucial function in which the APN recognition mode is highly conserved 29 Moreover the specificity of APN with the recognition structure in the S protein is linked to the structure of the APN N linked glycan and fusion with the RBD 1 2 turn In addition the CoVs tyrosine and tryptophan residues are critical in forming the TGEV RBD APN structure HCoV 229E does not have a tyro sine in its RBD 1 2 turn hence it recognizes the human APN that lacks this form of glycosylation 20 34 mean ing that HCoV 229E recognition of APN must be unique There have been some studies that determined the struc ture of a protruding tip for binding to small APN cavities in this human CoV The S proteins of PEDV and TGEV share high homology but they have different host prefer ences In addition PEDV has been verified to use a dif ferent receptor recognition model compared with TGEV PRCoV and HCoV NL63 The N terminal region in the PEDV S1 domain binds to sugars which are regarded as its coreceptor 20 Sialic Acid Promotes TGEV and PEDV Binding to Host Cell Receptors In addition to binding to defined protein receptors some CoVs show a sialic acid binding activity At present CoVs in and CoV have developed variant sialic acid binding activities 35 According to current re search 3 types of sugars have been characterized as recep tors or coreceptors for CoV entry into host cells 5 N acetylneuraminic acid Neu5Ac 5 N glycolylneuramin ic acid Neu5Gc and 5 N acetyl 9 O acetyl neuraminic acid Neu5 9Ac2 36 Recognition of sugars as co re ceptors of TGEV and PEDV seems to be a strategy by which these viruses adapt to the living organism meaning that TGEV and PEDV bind to sialic acid to survive under unfavorable intestinal tract conditions TGEV was first described to have a sialic acid binding activity in 1996 37 The sialic acid binding activity re sides in the N terminal portion of the S1 subunit that has been linked to the enteropathogenicity of TGEV and that is absent from the S protein of PRCoV The sialic acid preferentially recognized by TGEV is N glycolylneur aminic acid Neu5Ac 37 The spike protein has a tri meric structure and retains its sialic acid binding activity in soluble forms of the protein 38 39 TGEV recognizes and binds the sugar moieties of glycoconjugates that are highly O glycosylated which promotes binding but is not sufficient for initiation of infection It is believed that abundant sialic acid in mucins aids TGEV to penetrate the mucus layer and then to get access to pAPN on the surface of the intestinal epithelial cells Thus the efficien cy of infection can be enhanced under unfavorable condi tions Binding to pAPN and sialic acid are two indepen dent processes Interestingly binding to pAPN is more efficient in the absence of sialic acid 40 There are few studies on the binding of PEDV to sialic acids PEDV has been proven to have the ability to bind sialic acids Neu5Ac pr