Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

LDL receptor in alphavirus entry: structural analysis and implications for antiviral therapy.

Various low-density lipoprotein receptors (LPRs) have been identified as entry factors for alphaviruses, and structures of the corresponding virion-receptor complexes have been determined. Here, we analyze the similarities and differences in the receptor binding modes of multiple alphaviruses to understand their ability to infect a wide range of hosts. We further discuss the challenges associated with the development of broad-spectrum treatment strategies against a diverse range of alphaviruses.

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Untangling the Evolution of the Receptor-Binding Motif of SARS-CoV-2.

The spike protein determines the host-range specificity of coronaviruses. In particular, the Receptor-Binding Motif in the spike protein from SARS-CoV-2 contains the amino acids involved in molecular recognition of the host Angiotensin Converting Enzyme 2. Therefore, to understand how SARS-CoV-2 acquired its capacity to infect humans it is necessary to reconstruct the evolution of this important motif. Early during the pandemic, it was proposed that the SARS-CoV-2 Receptor-Binding Domain was acquired via recombination with a pangolin infecting coronavirus. This proposal was challenged by an alternative explanation that suggested that the Receptor-Binding Domain from SARS-CoV-2 did not originated via recombination with a coronavirus from a pangolin. Instead, this alternative hypothesis proposed that the Receptor-Binding Motif from the bat coronavirus RaTG13, was acquired via recombination with an unidentified coronavirus. And as a consequence of this event, the Receptor-Binding Domain from the pangolin coronavirus appeared as phylogenetically closer to SARS-CoV-2. Recently, the genomes from coronaviruses from Cambodia (bat_RShST182/200) and Laos (BANAL-20-52/103/247) which are closely related to SARS-CoV-2 were reported. However, no detailed analysis of the evolution of the Receptor-Binding Motif from these coronaviruses was reported. Here we revisit the evolution of the Receptor-Binding Domain and Motif in the light of the novel coronavirus genome sequences. Specifically, we wanted to test whether the above coronaviruses from Cambodia and Laos were the source of the Receptor-Binding Domain from RaTG13. We found that the Receptor-Binding Motif from these coronaviruses is phylogenetically closer to SARS-CoV-2 than to RaTG13. Therefore, the source of the Receptor-Binding Domain from RaTG13 is still unidentified. In accordance with previous studies, our results are consistent with the hypothesis that the Receptor-Binding Motif from SARS-CoV-2 evolved by vertical inheritance from a bat-infecting population of coronaviruses.

Structural basis for raccoon dog receptor recognition by SARS-CoV-2.

Since the COVID-19 outbreak, raccoon dogs have been suggested as a potential intermediary in transmitting SARS-CoV-2 to humans. To understand their role in the COVID-19 pandemic and the species barrier for SARS-CoV-2 transmission to humans, we analyzed how their ACE2 protein interacts with SARS-CoV-2 spike protein. Biochemical data showed that raccoon dog ACE2 is an effective receptor for SARS-CoV-2 spike protein, though not as effective as human ACE2. Structural comparisons highlighted differences in the virus-binding residues of raccoon dog ACE2 compared to human ACE2 (L24Q, Y34H, E38D, T82M, R353K), explaining their varied effectiveness as receptors for SARS-CoV-2. These variations contribute to the species barrier that exists between raccoon dogs and humans regarding SARS-CoV-2 transmission. Identifying these barriers can help assess the susceptibility of other mammals to SARS-CoV-2. Our research underscores the potential of raccoon dogs as SARS-CoV-2 carriers and identifies molecular barriers that affect the virus's ability to jump between species.

Computational methods in the analysis of SARS-CoV-2 in mammals: A systematic review of the literature.

SARS-CoV-2 is an enveloped RNA virus that causes severe respiratory illness in humans and animals. It infects cells by binding the Spike protein to the host's angiotensin-converting enzyme 2 (ACE2). The bat is considered the natural host of the virus, and zoonotic transmission is a significant risk and can happen when humans come into close contact with infected animals. Therefore, understanding the interconnection between human, animal, and environmental health is important to prevent and control future coronavirus outbreaks. This work aimed to systematically review the literature to identify characteristics that make mammals suitable virus transmitters and raise the main computational methods used to evaluate SARS-CoV-2 in mammals. Based on this review, it was possible to identify the main factors related to transmissions mentioned in the literature, such as the expression of ACE2 and proximity to humans, in addition to identifying the computational methods used for its study, such as Machine Learning, Molecular Modeling, Computational Simulation, between others. The findings of the work contribute to the prevention and control of future outbreaks, provide information on transmission factors, and highlight the importance of advanced computational methods in the study of infectious diseases that allow a deeper understanding of transmission patterns and can help in the development of more effective control and intervention strategies.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Binding Evolution of the Dengue Virus Envelope Against DC-SIGN: A Combined Approach of Phylogenetics and Molecular Dynamics Analyses Over 30 Years of Dengue Virus in Brazil.

The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Variation in the affinity of three representative avian adenoviruses for the cellular coxsackievirus and adenovirus receptor.

According to previous studies, three representative avian adenoviral strains utilize coxsackievirus-adenovirus receptor (CAR) as a receptor and seem to exhibit diverse binding affinities and modes. Thus, further revealing the exact molecular mechanism underlying the interaction between different FAdVs and the attachment receptor CAR is necessary. In this study, we successfully solved the crystal structure of the FAdV-4 fiber1 knob at 1.6 Šresolution. The interaction between the fibre knob and different domains of CAR was verified by confocal microscopy, coimmunoprecipitation and surface plasmon resonance (SPR) analysis. The fibre knobs of the three representative fowl adenoviruses specifically recognized CAR domain 1 (D1), but the recognition of CAR domain 2 (D2) by chicken embryo lethal orphan (CELO) strains was weak. These results provide insights into the differences in adenovirus‒host cell interactions and have important implications for the exploration of viral invasion mechanisms.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Predicting virus Fitness: Towards a structure-based computational model.

Predicting the impact of new emerging virus mutations is of major interest in surveillance and for understanding the evolutionary forces of the pathogens. The SARS-CoV-2 surface spike-protein (S-protein) binds to human ACE2 receptors as a critical step in host cell infection. At the same time, S-protein binding to human antibodies neutralizes the virus and prevents interaction with ACE2. Here we combine these two binding properties in a simple virus fitness model, using structure-based computation of all possible mutation effects averaged over 10 ACE2 complexes and 10 antibody complexes of the S-protein (∼380,000 computed mutations), and validated the approach against diverse experimental binding/escape data of ACE2 and antibodies. The ACE2-antibody selectivity change caused by mutation (i.e., the differential change in binding to ACE2 vs. immunity-inducing antibodies) is proposed to be a key metric of fitness model, enabling systematic error cancelation when evaluated. In this model, new mutations become fixated if they increase the selective binding to ACE2 relative to circulating antibodies, assuming that both are present in the host in a competitive binding situation. We use this model to categorize viral mutations that may best reach ACE2 before being captured by antibodies. Our model may aid the understanding of variant-specific vaccines and molecular mechanisms of viral evolution in the context of a human host.

Biophysical evolution of the receptor-binding domains of SARS-CoVs.

With hundreds of coronaviruses (CoVs) identified in bats that can infect humans, it is essential to understand how CoVs that affected the human population have evolved. Seven known CoVs have infected humans, of which three CoVs caused severe disease with high mortalities: severe acute respiratory syndrome (SARS)-CoV emerged in 2002, Middle East respiratory syndrome-CoV in 2012, and SARS-CoV-2 in 2019. SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor-binding domain (RBD) of spike protein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor-binding motif that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that, despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry toward ACE2 compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse receptor-binding motifs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrant further optimization of protein subunit vaccines that use RBD as an antigen by inserting stabilizing mutations or formulation screening.

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients.

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.

Exogenous Chemicals Impact Virus Receptor Gene Transcription: Insights from Deep Learning.

Despite the fact that coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been disrupting human life and health worldwide since the outbreak in late 2019, the impact of exogenous substance exposure on the viral infection remains unclear. It is well-known that, during viral infection, organism receptors play a significant role in mediating the entry of viruses to enter host cells. A major receptor of SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2). This study proposes a deep learning model based on the graph convolutional network (GCN) that enables, for the first time, the prediction of exogenous substances that affect the transcriptional expression of the ACE2 gene. It outperforms other machine learning models, achieving an area under receiver operating characteristic curve (AUROC) of 0.712 and 0.703 on the validation and internal test set, respectively. In addition, quantitative polymerase chain reaction (qPCR) experiments provided additional supporting evidence for indoor air pollutants identified by the GCN model. More broadly, the proposed methodology can be applied to predict the effect of environmental chemicals on the gene transcription of other virus receptors as well. In contrast to typical deep learning models that are of black box nature, we further highlight the interpretability of the proposed GCN model and how it facilitates deeper understanding of gene change at the structural level.

Biomimetic SARS-CoV-2 Spike Protein Nanoparticles.

COVID-19 is an infectious respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus contains a crucial coat protein that engages with target cells via a receptor binding domain (RBD) on its spike protein. To better study the RBD and its therapeutic opportunities, we genetically engineered a simple fusion with a thermo-responsive elastin-like polypeptide (ELP). These fusions express in Escherichia coli at a high yield in the soluble fraction and were easily purified using ELP-mediated phase separation (79 mg/L culture). Interestingly, they assembled peptide-based nanoparticles (Rh = 71.4 nm), which was attributed to oligomerization of RBDs (25.3 kDa) counterbalanced by steric stabilization by a soluble ELP (73.4 kDa). To investigate their biophysical properties, we explored the size, shape, and binding affinity for the human angiotensin-converting enzyme 2 (hACE2) and cellular uptake. Biomimetic nanoparticles such as these may enable future strategies to target the same cells, tissues, and cell-surface receptors as those harnessed by SARS-CoV-2.

Excavating phytochemicals from plants possessing antiviral activities for identifying SARS-CoV hemagglutinin-esterase inhibitors by diligent computational workflow.

Coronaviruses (CoVs) belong to a group of RNA viruses that cause diseases in vertebrates including. Newer and deadlier than SARS CoV-2 are sought to appear in future for which the scientific community must be prepared with the strategies for their control. Spike protein (S-protein) of all the CoVs require angiotensin-converting enzyme2 (ACE2), while CoVs also require hemagglutinin-acetylesterase (HE) glycoprotein receptor to simultaneously interact with O-acetylated sialic acids on host cells, both these interactions enable viral particle to enter host cell leading to its infection. Target inhibition of viral S-protein and HE glycoprotein receptor can lead to a development of therapy against the SARS CoV-2. The proposition is to recognize molecules from the bundle of phytochemicals of medicinal plants known to possess antiviral potentials as a lead that could interact and mask the active site of, HE glycoprotein which would ideally bind to O-acetylated sialic acids on human host cells. Such molecules can be addressed as 'HE glycoprotein blockers'. A library of 110 phytochemicals from Withania somnifera, Asparagus racemosus, Zinziber officinalis, Allium sativum, Curcuma longa and Adhatoda vasica was constructed and was used under present study. In silico analysis was employed with plant-derived phytochemicals. The molecular docking, molecular dynamics simulations over the scale of 1000 ns (1 µs) and ADMET prediction revealed that the Withania somnifera (ashwagandha) and Asparagus racemosus (shatavari) plants possessed various steroidal saponins and alkaloids which could potentially inhibit the COVID-19 virus and even other CoVs targeted HE glycoprotein receptor.Communicated by Ramaswamy H. Sarma.

Host range and structural analysis of bat-origin RshSTT182/200 coronavirus binding to human ACE2 and its animal orthologs.

Bat-origin RshSTT182 and RshSTT200 coronaviruses (CoV) from Rhinolophus shameli in Southeast Asia (Cambodia) share 92.6% whole-genome identity with SARS-CoV-2 and show identical receptor-binding domains (RBDs). In this study, we determined the structure of the RshSTT182/200 receptor binding domain (RBD) in complex with human angiotensin-converting enzyme 2 (hACE2) and identified the key residues that influence receptor binding. The binding of the RshSTT182/200 RBD to ACE2 orthologs from 39 animal species, including 18 bat species, was used to evaluate its host range. The RshSTT182/200 RBD broadly recognized 21 of 39 ACE2 orthologs, although its binding affinities for the orthologs were weaker than those of the RBD of SARS-CoV-2. Furthermore, RshSTT182 pseudovirus could utilize human, fox, and Rhinolophus affinis ACE2 receptors for cell entry. Moreover, we found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182 pseudovirus. Taken together, these findings indicate that RshSTT182/200 can potentially infect susceptible animals, but requires further evolution to obtain strong interspecies transmission abilities like SARS-CoV-2.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.