key: cord-290851-1e5e033r authors: Gerlier, Denis title: Emerging zoonotic viruses: new lessons on receptor and entry mechanisms date: 2011-06-12 journal: Curr Opin Virol DOI: 10.1016/j.coviro.2011.05.014 sha: doc_id: 290851 cord_uid: 1e5e033r Viruses enter the host cell by binding cellular receptors that allow appropriate delivery of the viral genome. Although the horizontal propagation of viruses feeds the continuous emergence of novel pathogenic viruses, the genetic variation of cellular receptors can represent a challenging barrier. The SARS coronavirus, henipaviruses and filoviruses are zoonotic RNA viruses that use bats as their reservoir. Their lethality for man has fostered extensive research both on the cellular receptors they use and their entry pathways. These studies have allowed new insights into the diversity of the molecular mechanisms underlying both virus entry and pathogenesis. Mankind is under a permanent threat from novel pathogens qualified as emerging [1,2 ,3] . Here I review the receptors and mode of entry of three emerging zoonotic viruses, responsible for rare but deadly diseases, whose natural reservoir is the bat: severe acute respiratory syndrome coronavirus (SARS-CoV), Hendra (HeV), Nipah (NiV), Ebola (EboV), and Marburg (MarV) viruses. SARS-CoV has a 30 kb positive RNA genome and the crown-like shape typical of the Coronavidae. A regular array of viral spike glycoprotein (S) trimers constitutes the viral envelope. S mediates binding to the cellular receptor Angiotensin Converting Enzyme 2 (ACE2) [4 ] , and ensures the viral-cell membrane fusion that allows virus entry. As an ectometalloprotease with monocarboxypeptidase activity, ACE2 cleaves the vasoconstrictor Angiotensin II octapeptide into the vasodilatator Ang1-7 heptapeptide. ACE2 protects the heart, lung and kidney from deleterious vasoconstriction and prevents the onset of an acute respiratory distress syndrome [4 ,5-8] . The tissue distribution of ACE2 (pneumocytes I and II, lung epithelium progenitor cells, small intestine enterocytes, kidney, heart cardiomyocytes and endothelium) mostly correlates with the known replication sites of SARS-CoV, and could explain the poor lung repair following SARS infection [4 ,9] . The ectopeptidase Type II transmembrane protease serine subfamily member 2 (TMPRSS2) was recently identified as a companion molecule of ACE2 [10 ,11 ,12 ] . TMPRSS2 is detected on the epithelium of the small intestine and respiratory tract, that is the major cell targets of SARS-CoV, but not on the endothelium, which is refractory to SARS-CoV infection [13, 14] . TMPRSS2 and ACE2 physically interact [10 ] . Only a few S proteins get cleaved by TMPRSS2 to allow a pH-and cathepsin-independent efficient entry of SARS-CoV [10 ,12 ] . TMPRSS2 cleaves S protein at sites distinct from those ascribed to trypsin and cathepsin L [12 ] . Upon contact of ACE2 with S protein, ACE2 is also cleaved by TMPRSS2 [10 ]. When expressed on opposing membranes, SARS-CoV S and the ACE2 + TMPRSS2 complex induce intercellular fusion [11 ] . However, newly expressed S proteins escape cleavage by TMPRSS2 allowing the production of virions decorated with uncleaved S [10 ,11 ,12 ], possibly because the tripartite association is prevented intracellularly. The present model of virus entry predicts the following ( Figure 1a ): SARS-CoV S protein binds to the ACE2 receptor via the concave S 424-494 region of the receptor binding site (RBD) that cradles over 17 nm 2 of the outer surface of the N-terminal lobe of the ACE peptidase domain, that is outside the enzymatic site [15 ] . This activates TMPRSS2 to cleave a few S proteins into fusioncompetent S1-S2 homodimers [10 ,11 ,12 ,16], which immediately undergo typical class I fusion protein structural changes [17] which permit the viral envelope to fuse with the plasma membrane. S1-S2 heterodimers are probably too unstable to be incorporated into infectious virus particles [16, 18] . Moreover, activated TMPRSS2, and possibly ADAM17/TACE (TNFa converting enzyme) [4 ,19] , concomitantly cleave ACE2. This results in the massive shedding of ACE2 ectodomains [10 ], probably due to amplification of the constitutive pathway [5] . ACE2 shedding is not required for SARS-COV entry [5, 20] , but is probably responsible for the associated major lung failure. Indeed, soluble S both induces ACE2 shed-ding and worsens the clinical signs of SARS [21] . In humans and according to virus strain diversity, S/ACE2 affinity correlates with the efficacy of virus entry, the level of ACE2 cleavage and the intensity of the pathology. Furthermore, the highest affinity is associated with interhuman transmission [20, 22 ] (Figure 1b) . Antibodies targeting the S binding site on ACE2 strongly inhibit viral infection [23] . The correlation between S/ACE2 affinity and SARS-CoV pathogenicity extends to the host range for other mammal species (except bats) in determining whether a particular ACE2 protein can act as a receptor for SARS-CoV or not (including among bats) ( NiV and HeV constitute the Henipavirus genus of the Paramyxoviridae family and are responsible for fatal respiratory and neurological diseases. Their non-segmented negative strand RNA genomes code for two envelope glycoproteins. The fusion protein F is synthesized as a precursor maturated into a functional F1-F2 heterodimer by cathepsin L via a clathrin-mediated recycling endosomal pathway [28, 29] . The attachment protein G is a tetramer consisting of two disulfide bridged dimers. Like the morbillivirus H and parainfluenzae HN proteins, its Cterminal globular head is folded into a six b-sheet blade propeller surmounting a stalk, transmembrane region and cytosolic tail. The sugar-free b1-b6 dimer interface is 28 Virus Entry conserved. The two heads rotate relative to each other by 08, 638 and 30-408 for henipavirus G, measles virus H and HN, respectively, while the buried area is 9-10 nm 2 EphA4 NiV-G and HeV-G for ephrinB2/B3 correlates with the efficiency of virus entry [48, 51] . Interestingly, whereas NiV G and F induce fusion of cells expressing ephrinB2/B3, NiV preferentially enters after internalization via macropinocytosis [52 ] , though acidic pH is not required [52 ,53] . Virus entry, but not membrane fusion, is inoperative when the cytosolic tail of ephrinB2 has its PDZ-binding motif deleted or Tyr 304 mutated [52 ] . These two motifs recruit Grb4 and the P21-activated kinase 1 (Pak1)/CdC42/Rac1 complex that govern macropinocytosis [33 , 54,55,56 ,57-59] . The need for macropinocytosis while the fusion machinery is operative at the cell surface is puzzling. Macropinocytosis occurs very rapidly upon contact [60] and could be faster than the fusion step but then macropinocytosis inhibitors would not be expected to prevent virus entry. Several hypotheses can be proposed: (i) fusion requires a specific Ca ++ [61] and/or Na + ionic environment as documented for Semliki Forest virus [62] . (ii) NiV replication requires a specific conditioning of the cytoplasm induced by contacting ephrinB2/B3. (iii) The nucleocapsid needs to reach a particular cytoplasmic location deeper in the cells, more favorable for viral polymerase activity. The latter would not be unprecedented since forced rerouting of virus normally entering by fusion at the cell surface into the endocytic pathway results in hampered infectivity as shown for pseudotyped measles virus and lentivectors [63] [64] [65] . Filoviruses: an elusive receptor The Filoviridae EboV and MarV cause severe hemorrhagic fevers and septic-like shock in humans [66] . Their non-segmented negative RNA genomes code for the envelope glycoprotein GP which ensures both attachment to a (still elusive) cellular receptor and membrane fusion. GP is cleaved by a furin-like protease into mature GP1-GP2 heterodimers [67] . Curiously, mutation of the furin-cleavage site does not abolish GP-mediated virus entry due to alternative cleavage [68] . GP is heavily glycosylated with sugar moieties recognized by LESCtin and DC-SIGN/R lectins that can enhance but not mediate infection [69] [70] [71] [72] . This high glycan content shields MHC class I and b-integrin from antibody recognition [73, 74] , a finding that explains the previously reported apparent downregulation of the latter [75] . A cellular receptor of glycoprotein nature is predicted on the basis of saturable binding of soluble GP [76] and loss of binding after protease, periodate or tunicamycin treatment [76, 77] . In infected animals, the virus disseminates in many tissues [66] . The EboV receptor is stocked in trans-Golgi network membranes in all cell types including the non-permissive T and B lymphocytes. It is exported to the cell surface upon cell adhesion and internalized via a microtubule-dependent and actin-dependent pathway, respectively [78 ,79 ] . EboV and MarV GP cross-compete for binding suggesting the use of a common receptor [76, 80] . However, 3 out of 4 key lysines (at positions 114,115 and 140) defining the receptor binding region (RBR) of EboV GP1 [76] are not conserved in MarV GP1 [81 ] . The structure of a soluble trimeric form of GP1-GP2 reveals a GP1-based chalice form, lined by the RBR. The fusion competent GP2 trimers cradle the chalice stem, with the internal fusion peptide flanked by two bsheets. The RBR is mostly shielded by a glycan cap and a mucin-like domain [82 ], the cleavage of which by cathepsins strongly enhances GP1-GP2 binding to the cell surface [76, 83] . However, lowering the pH neither allows EboV entry at the cell surface, nor cell-cell fusion by mucin-deleted GP1-GP2, and the GP/receptor interaction is stable at acidic pH [76, 77, 84 ] . In effect, EboV mostly enters by macropinocytosis with a requirement for lipid rafts, the Na + /H + exchanger, Pak 1, Rac1, Rab5, Rab7, RhoC GTPase and the vacuole closure protein C-terminal binding protein 1 of E1A, CtBP/BARS [59,85 ,86 ,87-89] . Constitutive macropinocytosis in dendritic cells and macrophages fits with their permissiveness to EboV infection [90, 91] . Activation of Ax1 enhances both macropinocytosis and EboV entry [92] although the latter may be mediated by serum Gas6, which was recently reported to mediate non-specific entry for several enveloped viruses [93] . The EboV (and MarV) entry process lasts for about 1 h [94, 95] and can be schematized as follows ( Figure 3) : Firstly, (i) EboV attaches to the cells via the GP1/GP2 interaction with DC-SIGN/R and/or LECStin and is (ii) immediately internalized by constitutive and/or virus-contact-induced macropinocytosis. (iii) After 30 min of endocytic trafficking, EboV reaches a late endosomal compartment, where (iv) the resident cathepsin B cleaves off the mucin-like domain [83,84 ,96] to (v) expose GP1's RBR so that the putative receptor can be recruited; then, (vi) a late pH-dependent activation step of the mucindeleted GP1/GP2 complex triggers the fusion activity of GP2, possibly via the reduction of a disulfide bridge [84 ] . In conclusion, several lessons can be taken home. These two papers demonstrate the intracellular pool of RBR binding protein of EboV that can be exported to the cell surface upon cell adhesion including in lymphocyte, which are refractory to EboV infection probably because they poorly able to prime GP. Emerging and reemerging diseases: a historical perspective Emerging infections: a perpetual challenge An historical overview that allows easy understanding of the ''emerging infection Global trends in emerging infectious diseases Trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters A review describing known physiological functions of ACE2 that allow the understanding of its key role in the severe lung injury induced by SARS-CoV Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia Angiotensinconverting enzyme 2 protects from severe acute lung failure of nipah virus in an experimentally infected cat Nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis Structural and biophysical characterization of the ephb4*ephrinb2 proteinprotein interaction and receptor specificity Crystal structure of an eph receptor-ephrin complex Functional studies of hostspecific ephrin-b ligands as henipavirus receptors A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection Feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine A quantitative and kinetic fusion protein-triggering assay can discern distinct steps in the nipah virus membrane fusion cascade Nipah virus entry can occur by macropinocytosis Simulating henipavirus multicycle replication in a screening assay leads to identification of a promising candidate for therapy The sh2/sh3 adaptor grb4 transduces b-ephrin reverse signals Tyrosine phosphorylation of transmembrane ligands for eph receptors Virus entry by macropinocytosis A useful review describing the molecular machinery of macropinocytosis and defining molecular criteria required for validating the use of this entry pathway by viruses Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake Subversion of ctbp1-controlled macropinocytosis by human adenovirus serotype 3 Dendritic cell function at low physiological temperature Tpcs: endolysosomal channels for ca2+ mobilization from acidic organelles triggered by naadp Effects of monovalent cations on semliki forest virus entry into bhk-21 cells Chimeric measles viruses with a foreign envelope Stable transduction of quiescent t cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity Ebola haemorrhagic fever Processing of the ebola virus glycoprotein by the proprotein convertase furin Endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function A novel mechanism for lsectin binding to ebola virus surface glycoprotein through truncated glycans Different potential of c-type lectin-mediated entry between marburg virus strains Lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus Interactions of lsectin and dc-sign/dc-signr with viral ligands: differential ph dependence, internalization and virion binding Steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein Ebolavirus glycoprotein gp masks both its own epitopes and the presence of cellular surface proteins Downregulation of beta1 integrins by ebola virus glycoprotein: implication for virus entry The primed ebolavirus glycoprotein (19-kilodalton gp1,2): sequence and residues critical for host cell binding Soluble GP and mutagenesis rationally designed from prefusion GP 3D structure allowed the detailed delineation of the RBR of EboV GP1 A system for functional analysis of ebola virus glycoprotein Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry Emerging zoonotic viruses: new lessons on receptor and entry mechanisms Gerlier 33 www.sciencedirect.com Current Opinion in Virology Cell adhesion promotes ebola virus envelope glycoprotein-mediated binding and infection Conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events Structure of the ebola virus glycoprotein bound to an antibody from a human survivor A first structure of prefusion EboV GP1-GP2 heterodimer showing the mucin-like domain covering the RBR site and location of GP2 at the basis of the GP1 chalice fold Proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity First demonstration for a two-step activation of EboV GP occurring in the endosomal pathway using chemical inhibitors, RNA silencing, and biochemical cleavage by cathepsins Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner Using Ebolavirus like particles made of GP and VP40, a close mimic of the filamentous EboV, infectious EboV and dedicated molecular tools and chemical inhibitors, entry by macropinocytosis was demonstrated by these two papers Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes Rho gtpases modulate entry of ebola virus and vesicular stomatitis virus pseudotyped vectors The closure of pak1-dependent macropinosomes requires the phosphorylation of ctbp1/bars Macropinocytotic uptake and infection of human epithelial cells with species b2 adenovirus type 35 Constitutive macropinocytosis allows tap-dependent major histocompatibility complex class i presentation of exogenous soluble antigen by bone marrow-derived dendritic cells Class i mhc presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages The tyro3 receptor kinase axl enhances macropinocytosis of zaire ebolavirus The soluble serum protein gas6 bridges virion envelope phosphatidylserine to the tam receptor tyrosine kinase axl to mediate viral entry Phosphoinositide-3 kinase-akt pathway controls cellular entry of ebola virus Come in and take your coat off-how host cells provide endocytosis for virus entry Endosomal proteolysis of the ebola virus glycoprotein is necessary for infection I apologize for numerous works not cited in this review because of space constraint. I thank R. Buckland, V. Volchkov and B. Horvat for their proof reading of the manuscript and useful comments. This work would not have been possible without the support from the Centre National de la Recherche Scientifique and 08_AFF-IDP grant from the Agence Nationale de la Recherche.