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71 Springer International Publishing AG part of Springer Nature 2018 E B ttcher Friebertsh user et al eds Activation of Viruses by Host Proteases https doi org 10 1007 978 3 319 75474 1 4 Priming Time How Cellular Proteases Arm Coronavirus Spike Proteins Markusuni00A0Hoffmann Heikeuni00A0Hofmann Winkler anduni00A0Stefanuni00A0P hlmann Abstract Coronaviruses are enveloped RNA viruses that infect mammals and birds Infection of humans with globally circulating human coronaviruses is associated with the common cold In contrast transmission of animal coronaviruses to humans can result in severe disease The severe acute respiratory syndrome SARS and the Middle East respiratory syndrome MERS are responsible for hundreds of deaths in Asia and the Middle East respectively and are both caused by members of the genus Betacoronavirus SARS CoV and MERS CoV that were zoonotically transmitted from an animal host to humans At present neither vaccines nor specif ic treatment is available to combat coronavirus infection in humans and novel antiviral strategies are urgently sought The viral spike pro tein S mediates the f irst essential step in coronavirus infection viral entry into target cells For this the S protein critically depends on priming by host cell proteases and the responsible enzymes are potential targets for antiviral inter vention Recent studies revealed that the endosomal cysteine protease cathepsin L and the serine proteases furin and TMPRSS2 prime the S proteins of SARS CoV and MERS CoV and provided evidence that successive S protein cleavage at two sites is required for S protein priming Moreover mechanisms that control protease choice were unraveled and insights were obtained into which enzyme promotes viral spread in the host Here we will provide basic information on S protein function and proteolytic priming and we will then discuss recent prog ress in our understanding of the priming of the S proteins of SARS CoV and MERS CoV M Hoffmann H Hofmann Winkler S P hlmann Infection Biology Unit German Primate Center G ttingen Germany e mail spoehlmann dpz eu 4 M H and H H W contributed equally to this work 72 4 1 Introduction Coronaviruses CoV belong to the Coronavirinae subfamily that forms along with the subfamily Torovirinae the virus family Coronaviridae within the order Nidovirales The Coronavirinae subfamily harbors four genera Fig uni00A04 1 Alpha Beta Gamma and Deltacoronavirus Adams and Carstens 2012 Woo etuni00A0al 2012 Coronaviruses are enveloped viruses that contain a single stranded RNA genome of positive polarity comprising roughly 30uni00A0kilobases The virus particles are spherical and with a diameter Canine CoV FIPV TGEV PEDV HCoV 229E HCoV NL63 Night heron CoV Munia CoV Bulbul CoV Porcine Deltacoronavirus Beluga whale CoV Pigeon CoV Duck CoV IBV 6 82 IBV Beaudette IBV M41 SARS CoV HKU 9 HKU 4 HKU 5 MERS CoV HKU 1 Bovine CoV HCoV OC43 MHV MHV 1 MHV ML 11 50 100 77 79 100 100 74 100 98 100 100 72 67 100 68 100 64100 100 100 97 75 100 100 Fig 4 1 Phylogenetic relationship among coronaviruses based on their spike glycoproteins The amino acid sequences of coronavirus spike glycoproteins representing all four genera Alpha uni03B1 Beta uni03B2 Gamma uni03B3 Deltacoronavirus uni03B4 within the Coronavirinae subfamily were aligned and utilized to generate a phylogenetic tree neighbor joining method Italicized numbers at the nodes indicate bootstrap values M Hof fmann et al 73 of 80 120uni00A0nm Belouzard etuni00A0al 2012 They contain the genome which is associated with the nucleoprotein NP forming a ribonucleoprotein complex RNP Belouzard etuni00A0al 2012 Depending on the virus three or four viral proteins are embedded in the viral envelope Membrane protein M envelope protein E and spike glycoprotein S are present in all coronaviruses while some members of the genus Betacoronavirus additionally contain a hemagglutinin esterase protein HE M and E are required for viral assembly Belouzard etuni00A0al 2012 HE promotes release of viruses from infected cells Vlasak etuni00A0al 1988 and the S protein which is in the focus of this review facili tates viral entry into target cells The S protein is also responsible for the corona like shape of these viruses in electron micrographs on the basis of which the name corona virus was coined Berry and Almeida 1968 Du etuni00A0al 2009 Coronaviruses infect a broad range of vertebrate hosts with alpha and betacoronavi ruses targeting different mammals while gamma and deltacoronaviruses mainly infect birds Breslin etuni00A0al 1999 Cavanagh etuni00A0al 2001 Jonassen etuni00A0al 2005 It is believed that coronaviruses of the genera Alpha and Betacoronavirus have emerged from bats while gamma and deltacoronaviruses seem to originate from birds Graham and Baric 2010 Woo etuni00A0al 2012 Coronavirus infection is mainly associated with respiratory and enteric diseases but depending on the virus can also lead to hepatic Lane and Hosking 2010 and neurologic manifestations Foley and Leutenegger 2001 Human coronaviruses HCoVs are known since 1965 when they were identif ied in patients suffering from the common cold Tyrrell and Bynoe 1965 Most of HCoVs known today HCoV 229E HCoV NL63 HCoV OC43 and HCoV HKU1 infect ciliated epithelia cells of the nasopharynx Afzelius 1994 Weiss and Navas Martin 2005 and cause self limiting upper respiratory tract diseases in immuno competent individuals with symptoms like headache sore throat and malaise being frequently observed In rare events infection can spread to the lower respiratory tract causing bronchiolitis bronchitis and pneumonia particularly in infants the elderly and immunocompromised individuals Masters and Perlman 2013 Within the last 20uni00A0years two novel HCoVs emerged that cause severe and fre quently fatal infections in humans Drosten etuni00A0al 2003 Lu etuni00A0al 2015 Reusken etuni00A0al 2016 Zaki etuni00A0al 2012 In 2002 the outbreak of severe acute respiratory syn drome coronavirus SARS CoV in Southern China and its subsequent worldwide spread was associated with roughly 8100 infections of which 10 took a fatal course with the elderly being mainly affected Peiris etuni00A0al 2003 In the aftermath of the SARS pandemic it has been revealed that bats harbor numerous SARS CoV related viruses as well as other coronaviruses that may be zoonotically transmitted to humans via intermediate hosts Hu etuni00A0 al 2015 Lu etuni00A0 al 2015 In 2012 the Middle East respiratory syndrome coronavirus MERS CoV another novel highly pathogenic coronavirus emerged in Saudi Arabia causing a SARS like disease Zaki etuni00A0al 2012 MERS CoV infection is associated with a case fatality rate of 35 WHO Health Organisation 2017 and comorbidities like diabetes mellitus chronic renal disease and hypertension constitute major risk factors for a lethal outcome of the disease Assiri etuni00A0al 2013 Like SARS CoV MERS CoV is a zoo notic virus originating from an animal reservoir dromedary camels Mohd etuni00A0al 2016 As the MERS epidemic is still ongoing there are concerns that 4 Coronavirus Spike Protein 74 human to human transmission which is very infrequent at present Alsolamy and Arabi 2015 might become more eff icient due to adaptive mutations in the viral genome Dudas and Rambaut 2016 Reusken etuni00A0al 2016 Coronaviruses also constitute a severe threat to animal health For instance por cine epidemic diarrhea coronavirus PEDV infects the epithelia of the small intes tine and causes villous atrophy resulting in diarrhea and severe dehydration Debouck and Pensaert 1980 Jung etuni00A0al 2006 The virus was f irst described in Europe in the 1970s and was originally not perceived as a major threat to animal health Debouck and Pensaert 1980 Pensaert and de 1978 Recently however highly virulent PEDV strains emerged that cause lethal infection in 80 100 of piglets and weight loss in adult pigs Debouck and Pensaert 1980 Lee 2015 PEDV spread can have severe consequences The introduction of PEDV in the USA resulted in major economic losses among pig farmers and a 10 decline in the American pig population Lee 2015 Li etuni00A0al 2012 Liu etuni00A0al 2016 Stevenson etuni00A0al 2013 As there are no effective vaccines or specif ic treatments available current containment strategies are mainly limited to rigorous disinfection routines Coronaviruses constitute a severe threat to animal and human health as discussed above and the development of antivirals is an important task Host cell factors required for coronavirus spread but dispensable for cellular survival are attractive targets since their blockade might suppress infection by several coronaviruses and might be associ ated with a high barrier against resistance development The viral S protein mediates the f irst step in coronavirus spread viral entry into target cells However the S protein is synthesized as an inactive precursor and requires cleavage by host cell proteases for conversion into an active form The cellular enzymes responsible constitute targets for antiviral intervention and recent studies provided important insights into their identity expression and target sites in the viral S protein Moreover novel mechanisms govern ing protease choice by coronaviruses have been uncovered The present manuscript will review and discuss these f indings focusing on SARS CoV and MERS CoV 4 2 The Coronavirus Spike Protein Viral Key foruni00A0Entry into theuni00A0Target Cell Domain organization The S protein of coronaviruses contains an N terminal signal peptide which primes the nascent polyprotein for import into the ER uni00A0In the ER the S protein is extensively modif ied with N linked glycans which may provide protec tion against neutralizing antibodies Walls etuni00A0al 2016b After passing the quality control mechanisms of the ER the S protein is transported to the site of viral bud ding the endoplasmic reticulum Golgi intermediate compartment ERGIC Homotrimers of the S protein for which atomic structures have recently been reported Kirchdoerfer etuni00A0al 2016 Walls etuni00A0al 2016a are incorporated into the viral membrane and mediate viral entry into target cells For this the S protein com bines two biological functions First its surface unit S1 binds to a specif ic receptor located at the surface of host cells and thereby determines cellular tropism and as a consequence viral pathogenesis Second the transmembrane unit S2 mediates fusion between the viral envelope and a target cell membrane Fig uni00A04 2 M Hof fmann et al 75 HR1 RBD Signal peptide Fusion peptide S1 Receptor binding S2 Fusion RBD Viral membrane R667 R797 HR2 Endodomain Transmembrane domain CN a b Fig 4 2 Domain organization and structure of the coronavirus spike glycoprotein a Schematic illustration of a coronavirus spike S glycoprotein consisting of the subdomains S1 and S2 At the N terminus of the S1 subdomain resides the signal peptide that allows for introduction of nascent S proteins into the host cells secretory pathway Additionally this subdomain harbors amino acid residues responsible for virus attachment to target cells receptor binding domain RBD The S2 subdomain contains the structural components of the membrane fusion machinery fusion peptide heptad repeats HR 1 and 2 anchors the S protein in the lipid envelope via the transmembrane domain and interacts with the viral ribonucleoprotein complex through its endodomain Location of the S1 S2 border and the S2uni2032 position is indicated by black triangles b 3D model of trimeric SARS CoV S protein amino acid residues 261 1058 schematically positioned on the outside of the viral envelope The protein structure ID 5WRG Gui etuni00A0al 2017 was downloaded from the RCSB Protein Data Bank and analyzed using the YASARA software www yasara org Krieger and Vriend 2014 Each S protein monomer is colored individually and the position of the RBD is indicated Further the locations of the arginines at the S1 S2 border R667 and S2uni2032 position R797 are highlighted 4 Coronavirus Spike Protein 76 Cellular receptors Coronaviruses use a broad range of receptors for entry into target cells Tableuni00A04 1 Alphacoronaviruses like HCoV 229E transmissible gastro enteritis coronavirus TGEV and porcine respiratory coronavirus PRCV engage the zinc metalloproteinase CD13 from their natural host as well as feline CD13 feCD13 as entry receptor Tresnan and Holmes 1998 with different residues in feCD13 being required for recognition by the respective coronaviral S proteins Tusell etuni00A0al 2007 Despite high amino acid sequence similarity within the S1 sub unit the S proteins of HCoV 229E and NL63 interact with different host cell recep tors namely CD13 Yeager etuni00A0 al 1992 and angiotensin converting enzyme 2 ACE2 Hofmann etuni00A0al 2005 Notably ACE2 is also employed by SARS CoV for entry Li etuni00A0al 2003 Wang etuni00A0al 2004 although the S protein of this betacoronavi rus and NL63 S share little sequence similarity Other members of the betacorona viruses use different entry receptors MERS CoV uses human dipeptidyl peptidase 4 DPP4 mouse hepatitis virus MHV interacts with carcinoembryonic antigen related cell adhesion molecule 1 CEACAM1 Dveksler etuni00A0al 1991 Williams etuni00A0al 1991 and neuraminic acid is used by bovine CoV and HCoV OC43 for attachment to cells Kunkel and Herrler 1993 Schultze etuni00A0 al 1991 Similarly sialic acid containing surface molecules serve as attachment factors or receptors for TGEV PEDV and avian infectious bronchitis virus IBV Cavanagh and Davis 1986 Deng etuni00A0al 2016 Krempl etuni00A0al 1997 Liu etuni00A0al 2015 Schultze etuni00A0al 1992 Structural insights into receptor choice The proteolytic priming of the viral S proteins is in the center of this review However priming and receptor binding can be intimately connected and structural analyses provide valuable explanations for coronavirus receptor specif icity Therefore structural aspects of S protein binding to its receptor will be brief ly discussed Binding to a receptor is mediated by a receptor binding domain RBD which is located in the surface unit S1 The S1 subunit generally consists of an N terminal NTD and a C terminal domain CTD Li 2012 which can serve as RBD either alone or in combination For most coro navirus analyzed the S1 NTD is responsible for binding to host cell glycans Krempl etuni00A0al 1997 Liu etuni00A0al 2015 Peng etuni00A0al 2012 Promkuntod etuni00A0al 2014 whereas the S1 CTD targets the a proteinaceous receptor Du etuni00A0al 2013 Godet etuni00A0al 1994 Hofmann etuni00A0al 2006 Lin etuni00A0al 2008 Liu etuni00A0al 2015 Mou etuni00A0al 2013 Wong etuni00A0 al 2004 All S1 CTD investigated so far are characterized by a core Table 4 1 Host cell receptors of selected alpha and betacoronaviruses Genus Virus Receptor Alphacoronavirus FIPV CD13 TGEV CD13 HCoV NL63 Angiotensin converting enzyme 2 ACE2 HCoV 229E CD13 Betacoronavirus SARS CoV Angiotensin converting enzyme 2 ACE2 MERS CoV Dipeptidyl peptidase 4 DPP4 HCoV OC43 N Acetyl 9 O acetyl neuraminic acid Neu5 9Ac2 MHV Carcinoembryonic antigen related cell adhesion molecule 1a CEACAM1a M Hof fmann et al 77 domain overlaid by an external region which directly contacts the receptor Li 2016 The S1 CTD of SARS S comprises a core of f ive sheets in antiparallel ori entation headed by a rather globular external region Li etuni00A0al 2005a in which amino acids N479 and T487 mediate high aff inity binding to ACE2 Li etuni00A0al 2005b The S protein of SARS CoV from palm civets a potential intermediate host Guan etuni00A0al 2003 Ksiazek etuni00A0al 2003 Rota etuni00A0al 2003 Song etuni00A0al 2005 Wu etuni00A0al 2005 harbors amino acids at positions 479 and 487 which preclude eff icient binding to human ACE2 Li 2008 and acquisition of mutations at these positions was suff icient for cross species transmission during the SARS epidemic Li 2008 Li etuni00A0al 2005b Qu etuni00A0al 2005 Song etuni00A0al 2005 Wu etuni00A0al 2011 2012 Within human ACE2 two lysine residues K31 and K353 are critical for SARS S binding Li 2008 Wu etuni00A0al 2011 2012 and an exchange to histidine at position 353 present in murine ACE2 renders this protein unsuitable for eff icient SARS S binding Li etuni00A0al 2004 2005b Similarly the rat homologue of ACE2 contains a glycosylated asparagine at position 82 which sterically blocks S protein interaction Frieman etuni00A0al 2012 Li etuni00A0al 2004 These f ind ings show that subtle variations within the S protein and its receptor can dramatically impact cross species transmission of coronaviruses The core domain of the S1 CTD in MERS S structurally resembles that of SARS S Chen etuni00A0al 2013 Lu etuni00A0al 2013 Wang etuni00A0al 2013 Yuan etuni00A0al 2017 but the extended core domains are different with the MERS S extended core consisting of antiparallel sheets forming a f lat surface which targets DPP4 Raj etuni00A0al 2013 The MERS S binding site on DPP4 is located within a propeller like structure con served in bat camel and human DPP4 Barlan etuni00A0al 2014 van etuni00A0al 2014 and MERS related CoV have been isolated from both bats and camels Alagaili etuni00A0al 2014 Annan etuni00A0al 2013 Haagmans etuni00A0al 2014 Lau etuni00A0al 2013 In contrast rodent DPP4 homologues are nonfunctional as MERS CoV receptors Cockrell etuni00A0al 2014 Coleman etuni00A0al 2014 Fukuma etuni00A0al 2015 Peck etuni00A0al 2015 Raj etuni00A0al 2014 proba bly due to steric hindrance due to a glycosylation in rodent DPP4 Peck etuni00A0al 2015 In a recent publication Yuan and colleagues analyzed trimeric MERS and SARS S proteins in their pre fusion conformation using single particle cryo electron microscopy Yuan etuni00A0al 2017 Their results revealed an unexpected f lexi bility of the respective RBDs in the lying state the RBDs are buried inside the trimer whereas in the standing state the RBDs are exposed for receptor interac tion Yuan etuni00A0al 2017 Hereby MERS S1 S2 trimers appeared with one or two of the RBDs in the standing conformation thus being able to contact DPP4 whereas SARS S trimers showed two or all three RBDs in the lying state thus being inca pable of receptor binding without further conformational change The f lexibility of the RBDs might therefore alleviate receptor interaction for subsequent virus entry Yuan etuni00A0al 2017 Finally it should be noted that the RBD constitutes the most important target for neutralizing antibodies Bonavia etuni00A0al 2003 Breslin etuni00A0al 2003 Godet etuni00A0al 1994 He etuni00A0al 2004 Kubo etuni00A0al 1994 Additionally sequence comparison of six HCoV S2 domains suggests that also the fusion peptide the HR1 domain and the central helix which are exposed at the surface of the stem region of S protein trimers can be targeted by neutralizing antibodies Yuan etuni00A0al 2017 Therefore the structural 4 Coronavirus Spike Protein 78 information discussed above not only provides insights into S protein receptor inter actions but also helps to understand how they can be inhibited by antibodies Du etuni00A0al 2008 Lan etuni00A0al 2015 Oh etuni00A0al 2014 Tai etuni00A0al 2017 Walls etuni00A0al 2016a Membrane fusion The transmembrane unit S2 harbors domains required for fusion between viral and host cell membrane including a fusion peptide and two heptad repeats HR1 and HR2 These elements are followed by a transmembrane TM domain and a C terminal intracytoplasmic tail Fig uni00A04 2 which plays a role in S pro tein sorting The HR domains consist of uni03B1 helices and their position and amino acid sequences are conserved among all groups within the coronavirus family de Groot etuni00A0al 1987 Membrane fusion commences with the insertion of the fusion peptide into the target cell membrane Subsequently the HR regions fold back onto each other resulting in the formation of a thermostable six helix bundle structure Bosch etuni00A0al 2003 Duquerroy etuni00A0al 2005 Lu etuni00A0al 2014 White and Whittaker 2016 As a consequen