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Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Significance Nipah and Hendra viruses are pathogenic zoonotic henipaviruses, transmitted from wildlife to humans, and potential epidemic threats. Zoonoses from wildlife hosts to humans require overcoming barriers that limit cross-species transmission. Successful usage of virus entry receptors represents a bottleneck to zoonosis. Conserved ephrins are recognized entry receptors for henipaviruses like Nipah virus. Here, we characterize the ephrin entry receptor usage of Cedar virus (CedV), a related henipavirus with unknown zoonotic potential that is nonpathogenic in animals known to be susceptible to Nipah and Hendra disease. We discovered that CedV utilizes 4 different ephrins, 3 of which are unique receptors for CedV, and determined that a single natural amino acid difference between human and mouse ephrin-A1 can dictate functional receptor usage., Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses (HNVs) causing respiratory illness and severe encephalitis in humans, with fatality rates of 50-100%. There are no licensed therapeutics or vaccines to protect humans. HeV and NiV use a receptor-binding glycoprotein (G) and a fusion glycoprotein (F) to enter host cells. HNV F and G are the main targets of the humoral immune response, and the presence of neutralizing antibodies is a correlate of protection against NiV and HeV in experimentally infected animals. We describe here two cross-reactive F-specific antibodies, 1F5 and 12B2, that neutralize NiV and HeV through inhibition of membrane fusion. Cryo-electron microscopy structures reveal that 1F5 and 12B2 recognize distinct prefusion-specific, conserved quaternary epitopes and lock F in its prefusion conformation. We provide proof-of-concept for using antibody cocktails for neutralizing NiV and HeV and define a roadmap for developing effective countermeasures against these highly pathogenic viruses.

Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers. IMPORTANCE Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion protein trimer. Structural analyses of the receptor binding hemagglutinin-neuraminidases of certain paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H heads on a leash long enough to adopt a triggering-permissive conformation but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a “leash,” suggesting a conserved triggering mechanism.

Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies viruses, use ubiquitous nectins 1 and 2. The negative-strand RNA virus measles virus (MeV) uses tissue-specific nectin-4, and the positive-strand RNA virus poliovirus uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C′C″, and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C′C″ loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C′C″ loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation and MeV-induced fusion. IMPORTANCE Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin requires only the BC and FG loops. However, we discovered that residues in the C′C″ loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

Wild-type measles virus (MV) strains use the signaling lymphocytic activation molecule (SLAM; CD150) and the adherens junction protein nectin-4 (poliovirus receptor-like 4 [PVRL4]) as receptors. Vaccine MV strains have adapted to use ubiquitous membrane cofactor protein (MCP; CD46) in addition. Recently solved cocrystal structures of the MV attachment protein (hemagglutinin [H]) with each receptor indicate that all three bind close to a hydrophobic groove located between blades 4 and 5 (β4-β5 groove) of the H protein β-propeller head. We used this structural information to focus our analysis of the functional footprints of the three receptors on vaccine MV H. We mutagenized this protein and tested the ability of individual mutants to support cell fusion through each receptor. The results highlighted a strong overlap between the functional footprints of nectin-4 and CD46 but not those of SLAM. A soluble form of nectin-4 abolished vaccine MV entry in nectin-4- and CD46-expressing cells but only reduced entry through SLAM. Analyses of the binding kinetics of an H mutant with the three receptors revealed that a single substitution in the β4-β5 groove drastically reduced nectin-4 and CD46 binding while minimally altering SLAM binding. We also generated recombinant viruses and analyzed their infections in cells expressing individual receptors. Introduction of a single substitution into the hydrophobic pocket affected entry through both nectin-4 and CD46 but not through SLAM. Thus, while nectin-4 and CD46 interact functionally with the H protein β4-β5 hydrophobic groove, SLAM merely covers it. This has implications for vaccine and antiviral strategies.

Antibody-mediated neutralization may interfere with the efficacy of measles virus (MV) oncolysis. To circumvent vector neutralization, we sought to exchange the envelope glycoproteins, hemagglutinin (H) and fusion (F), with those from the non-crossreactive Tupaia paramyxovirus (TPMV). To sustain efficient particle assembly, we generated hybrid glycoproteins with the MV cytoplasmic tails and the TPMV ectodomains. Hybrid F proteins that partially retained fusion function, and hybrid H proteins that retained fusion support activity, were generated. However, when used in combination, the hybrid proteins did not support membrane fusion. An alternative strategy was developed based on a hybrid F protein and a truncated H protein that supported cell-cell fusion. A hybrid virus expressing these two proteins was rescued, and was able to spread by cell fusion; however, it was only capable of producing minimal amounts of particles. Lack of specific interactions between the matrix and the H protein, in combination with suboptimal F-protein processing and inefficient glycoprotein transport in the rescue cells, accounted for inefficient particle production. Ultimately, this interferes with applications for oncolytic virotherapy. Alternative strategies for the generation of shielded MV are discussed.

We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and herpes simplex virus can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids.

International audience; Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

The measles virus entry system, consisting of attachment (hemagglutinin, H) and fusion proteins, operates by means of a variety of natural and targeted receptors; however, the mechanism that triggers fusion of the viral envelope with the plasma membrane is not understood. Here, we tested a model proposing that the two heads of an H dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface with additional intermolecular disulfide bonds prevented membrane fusion, an effect that was reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided that it engaged the H dimer at locations proximal to where the natural receptors bind and distal to the H-dimer interface. We discuss how receptors may force H-protein heads to switch partners and transmit the fusion-triggering signal.

The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1–E2 contacts, and receptor binding.

International audience; A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious.

The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.