Yanfang Zhang

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus–cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7u2009nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein–protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses.

Two Viruses to Bind Structural studies of two different H7N9 influenza viruses isolated from humans—A/Shanghai/1/2013 and A/Anhui/1/2013—which have different amino acid sequences in the receptor binding site, provide data indicating that the virus is in transition with respect to host adaptation. The Shanghai virus was one of the first isolated in humans that binds avian receptor glycans with high affinity, but binds poorly to human receptors. However, the later Anhui isolates can bind both avian and human receptors at high affinity. Shi et al. (p. 243, published online 5 September) show that four hydrophobic mutations contribute to acquisition of affinity for the human receptor by the virus hemagglutinin (HA) and confirm this effect in binding studies with virus particles. Further comparison of a mutant H7N9 A/Anhui/1/2013 HA with the bird flu H5N1 virus revealed the significance of some of the naturally occurring changes observed in circulating H7N9 viruses, which helps to explain how these viruses have been able to cause many severe human infections in a short time. Four amino acids in the H7N9 influenza virus binding site provide a hydrophobic environment for human receptors. An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor–binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln226) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu226). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu226 → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor–binding property.

Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a current global public health concern. The flavivirus envelope (E) glycoprotein is responsible for virus entry and represents a major target of neutralizing antibodies for other flaviviruses. Here, we report the structures of ZIKV E protein at 2.0xa0Å and in complex with a flavivirus broadly neutralizing murine antibody 2A10G6 at 3.0xa0Å. ZIKV-E resembles all the known flavivirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity inxa0vitro and neutralizes currently circulating ZIKV strains inxa0vitro and in mice. The E protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV.

Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.

Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka’s group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD). We captured two states of the RBD with receptor binding region either buried (lying state) or exposed (standing state), demonstrating an inherently flexible RBD readily recognized by the receptor. Further sequence conservation analysis of six human-infecting coronaviruses revealed that the fusion peptide, HR1 region and the central helix are potential targets for eliciting broadly neutralizing antibodies.

Structure and Receptor Binding Specificity of Hemagglutinin H13 from Avian Influenza A Virus H13N6

ABSTRACT Interspecies transmission (host switching/jumping) of influenza viruses is a key scientific question that must be addressed. In addition to the vigorous research on highly pathogenic avian influenza viruses (HPAIVs), studies of the mechanism of interspecies transmission of low-pathogenic avian influenza viruses (LPAIVs) could also provide insights into host tropism and virulence evolution. Influenza A viruses harboring hemagglutinin (HA) H13 (e.g., H13N6) are LPAIVs. In this study, soluble H13 HA glycoprotein was purified, and its receptor binding activity was characterized. The results revealed that H13 exclusively binds the avian α2-3-linked sialic acid receptor; no binding to the mammalian α2-6-linked sialic acid receptor was detected. Furthermore, the molecular basis of the H13 receptor binding specificity was revealed by comparative analysis of the crystal structures of both receptor-bound H13 and H5 HAs, which might be contributed by the hydrophobic residue V186. Work with an H13N186 mutant confirmed the importance of V186 in the receptor binding specificity of H13 HA, which shows that the mutant protein reduced the binding of an avian receptor analog but increased the binding of a human receptor analog. Detailed structural analysis also demonstrated that the conserved binding sites of the recently well-studied broadly neutralizing human monoclonal antibodies targeting the HA2 domain are found in H13. Our results expand our understanding of virulence evolution, receptor binding preference, and species tropism of the LPAIVs and HPAIVs.

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.

Significance Bunyaviruses are emerging zoonotic pathogens of public-health concern. Lack of structures for proteins on the viral membrane (“envelope”) surface limits understanding of entry. We describe atomic-level structures for the globular “head” of the envelope protein, glycoprotein N (Gn), from two members, severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV), of Phleboviruses genus in the bunyavirus family, and a structure of the SFTSV Gn bound with a neutralizing antibody Fab. The results show the folded Gn structure and define virus-specific neutralizing-antibody binding sites. Biochemical assays suggest that dimerization, mediated by conserved cysteines in the region (“stem”) connecting the Gn head with the transmembrane domain, is a general feature of bunyavirus envelope proteins and that the dimer is probably the olimeric form on the viral surface. Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae. Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4–5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

PILRα and PILRβ have a siglec fold and provide the basis of binding to sialic acid

Significance The paired immunoglobulin-like type 2 receptor α (PILRα) and β (PILRβ) are important surface molecules which, upon ligand binding, can deliver opposing signals to modulate the host immune responses. In this study, we elucidated the molecular basis on the ligand binding of PILRs by systematic structural and functional assays. Both PILRα and PILRβ show a typical siglec-like fold but exhibit variant binding avidities for sialic acid (SA). We further identified key residues responsible for SA binding and elucidated the atomic interaction details via a complex crystal structure. In conclusion, the SA recognition mechanism for the PILR receptors has been, for the first time to our knowledge, systematically investigated and clearly presented. Paired immunoglobulin-like type 2 receptor α (PILRα) and β (PILRβ) belong to the PILR family and are related to innate immune regulation in various species. Despite their high sequence identity, PILRα and PILRβ are shown to have variant sialic acid (SA) binding avidities. To explore the molecular basis of this interaction, we solved the crystal structures of PILRα and PILRβ at resolutions of 1.6 Å and 2.2 Å, respectively. Both molecules adopt a typical siglec fold but use a hydrophobic bond to substitute the siglec-specific disulfide linkage for protein stabilization. We further used HSV-1 glycoprotein B (gB) as a representative molecule to study the PILR–SA interaction. Deploying site-directed mutagenesis, we demonstrated that three residues (Y2, R95, and W108) presented on the surface of PILRα form the SA binding site equivalent to those in siglecs but are arranged in a unique linear mode. PILRβ differs from PILRα in one of these three residues (L108), explaining its inability to engage gB. Mutation of L108 to tryptophan in PILRβ restored the gB-binding capacity. We further solved the structure of this PILRβ mutant complexed with SA, which reveals the atomic details mediating PILR/SA recognition. In comparison with the free PILR structures, amino acid Y2 oriented variantly in the complex structure, thereby disrupting the linear arrangement of PILR residues Y2, R95, and W108. In conclusion, our study provides significant implications for the PILR–SA interaction and paves the way for understanding PILR-related ligand binding.

Putative Receptor Binding Domain of Bat-Derived Coronavirus HKU9 Spike Protein: Evolution of Betacoronavirus Receptor Binding Motifs

The suggested bat origin for Middle East respiratory syndrome coronavirus (MERS-CoV) has revitalized the studies of other bat-derived coronaviruses with respect to interspecies transmission potential. Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (betaCoV) that is phylogenetically affiliated with the same genus as MERS-CoV. The bat surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential to cross species barriers. The receptor binding domain (RBD) of the coronavirus spike (S) protein recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other betaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By using surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither SARS-CoV receptor ACE2 nor MERS-CoV receptor CD26. We further determined the atomic structure of HKU9-RBD, which as expected is composed of a core and an external subdomain. The core subdomain fold resembles those of other betaCoV RBDs, whereas the external subdomain is structurally unique with a single helix, explaining the inability of HKU9-RBD to react with either ACE2 or CD26. Via comparison of the available RBD structures, we further proposed a homologous intersubdomain binding mode in betaCoV RBDs that anchors the external subdomain to the core subdomain. The revealed RBD features would shed light on the evolution route of betaCoV.