Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.     

Repurposing of approved drugs with potential to interact with SARS-CoV-2 receptor

Respiratory transmission is the primary route of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Angiotensin I converting enzyme 2 (ACE2) is the known receptor of SARS-CoV-2 surface spike glycoprotein for entry into human cells. A recent study reported absent to low expression of ACE2 in a variety of human lung epithelial cell samples. Three bioprojects (PRJEB4337, PRJNA270632 and PRJNA280600) invariably found abundant expression of ACE1 (a homolog of ACE2 and also known as ACE) in human lungs compared to very low expression of ACE2. In fact, ACE1 has a wider and more abundant tissue distribution compared to ACE2. Although it is not obvious from the primary sequence alignment of ACE1 and ACE2, comparison of X-ray crystallographic structures show striking similarities in the regions of the peptidase domains (PD) of these proteins, which is known (for ACE2) to interact with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Critical amino acids in ACE2 that mediate interaction with the viral spike protein are present and organized in the same order in the PD of ACE1. In silico analysis predicts comparable interaction of SARS-CoV-2 spike protein with ACE1 and ACE2. In addition, this study predicts from a list of 1263 already approved drugs that may interact with ACE2 and/or ACE1 and potentially interfere with the entry of SARS-CoV-2 inside the host cells.     

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.     

Low expression of TMPRSS2–a SARS-CoV-2 internalization protease–associates with basal subtype of head and neck squamous cell carcinoma

SARS-CoV-2 is a single-stranded RNA virus that has caused the ongoing COVID-19 pandemic. ACE2 and other genes utilized by SARS-CoV-2 to enter human cells have been shown to express in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, their expression pattern in different subtypes has not been investigated. Hence, in the current study, we have analyzed the expression of ACE2, TMPRSS2 and FURIN in 649 HNSCC patients from two independent cohorts. Our analysis showed significantly lower expression of TMPRSS2 while significantly increased expression of ACE2 and FURIN in HPV-negative HNSCC. Comparison of expression of these genes in the three subtypes of HNSCC patients (basal, classical and inflamed/mesenchymal) showed no significant difference in the expression of ACE2 among the three subtypes; however, the basal subtype showed significantly reduced expression of TMPRSS2 but significantly increased expression of FURIN. Comparison of expression of these genes between the HPV-negative patients of basal subtype vs all others confirmed significantly lower expression of TMPRSS2 in HPV-negative patients of basal subtype as compared to all others. Our study shows that the different subtypes of HNSCC patients have different expression patterns of genes utilized by the SARS-CoV-2 to enter human cells, and hence, their susceptibility to SARS-CoV-2 may also be different. As the expression of TMPRSS2 is significantly lower in the HNSCC patients of the basal subtype, we predict that these patients would be less susceptible to SARS-CoV-2 infection than the patients of other subtypes. However, these findings need to be further validated.     

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.     

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.     

Nasopharyngeal microbiome reveals the prevalence of opportunistic pathogens in SARS-CoV-2 infected individuals and their association with host types

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is causing a severe global health emergency owing to its highly infectious nature. Although the symptoms of SARS-CoV-2 are well known but its impact on nasopharyngeal microbiome is poorly studied. The present cross-sectional study was intended to understand the perturbation in the nasopharyngeal microbiome composition within the infected (n = 63) and non-infected (n = 26) individuals using 16S rRNA gene based targeted amplicon sequencing and their association with host types and the prevalence of opportunistic pathogens at the stage of infection. The results confirmed that number of OTUs were significantly (p < 0.05) decreased in the SARS-CoV-2 infected individuals in comparison to non-infected individuals. Pairwise Wilcoxon test showed a significant (p < 0.05) increase in the abundance of Proteobacteria in infected individuals compared to non-infected ones and vice-versa for Fusobacteria and Bacteroidetes. Similarity percentage (SIMPER) analysis showed the increment in the abundance of opportunistic pathogens (Haemophilus, Stenotrophomonas, Acinetobacter, Moraxella, Corynebacterium 1, Gemella, Ralstonia, and Pseudomonas) involved in secondary infection. Furthermore, this study highlighted the microbial community structure of individuals within and across the families. In this study, we also performed the assesment of microbiome associated with host types (age and genders) and COVID-19 conditions (symptomatic and asymptomatic). The data suggested that the host types/conditions during the COVID-19 infection are potential factors in enrichment of specific bacterial communities in upper respiratory tract.     

Molecular dynamic and bioinformatic studies of metformin-induced ACE2 phosphorylation in the presence of different SARS-CoV-2 S protein mutations

The SARS-CoV-2 infection activates host kinases and causes high phosphorylation in both the host and the virus. There were around 70 phosphorylation sites found in SARS-CoV-2 viral proteins. Besides, almost 15,000 host phosphorylation sites were found in SARS-CoV-2-infected cells. COVID-19 is thought to enter cells via the well-known receptor Angiotensin-Converting Enzyme 2 (ACE2) and the serine protease TMPRSS2. Substantially, the COVID-19 infection doesn’t induce phosphorylation of the ACE2 receptor at Serin-680(s680). Metformin's numerous pleiotropic properties and extensive use in medicine including COVID-19, have inspired experts to call it the “aspirin of the twenty-first century”. Metformin's impact on COVID-19 has been verified in clinical investigations via ACE2 receptor phosphorylation at s680. In the infection of COVID-19, sodium-dependent transporters including the major neutral amino acid (B⁰AT1) is regulated by ACE2. The structure of B⁰AT1 complexing with the COVID-19 receptor ACE2 enabled significant progress in the creation of mRNA vaccines. We aimed to study the impact of the interaction of the phosphorylation form of ACE2-s680 with wild-type (WT) and different mutations of SARS-CoV-2 infection such as delta, omicron, and gamma (γ) on their entrance of host cells as well as the regulation of B⁰AT1by the SARS-CoV-2 receptor ACE2. Interestingly, compared to WT SARS-CoV-2, ACE2 receptor phosphorylation at s680 produces conformational alterations in all types of SARS-CoV-2. Furthermore, our results showed for the first time that this phosphorylation significantly influences ACE2 sites K625, K676, and R678, which are key mediators for ACE2-B⁰AT1 complex.     

The Drosophila melanogaster ACE2 ortholog genes are differently expressed in obesity/diabetes and aging models: Implications for COVID-19 pathology

The Spike glycoprotein of SARS-CoV-2, the virus responsible for coronavirus disease 2019, binds to its ACE2 receptor for internalization in the host cells. Elderly individuals or those with subjacent disorders, such as obesity and diabetes, are more susceptible to COVID-19 severity. Additionally, several SARS-CoV-2 variants appear to enhance the Spike-ACE2 interaction, which increases transmissibility and death. Considering that the fruit fly is a robust animal model in metabolic research and has two ACE2 orthologs, Ance and Acer, in this work, we studied the effects of two hypercaloric diets (HFD and HSD) and aging on ACE2 orthologs mRNA expression levels in Drosophila melanogaster. To complement our work, we analyzed the predicted binding affinity between the Spike protein with Ance and Acer. We show for the first time that Ance and Acer genes are differentially regulated and dependent on diet and age in adult flies. At the molecular level, Ance and Acer proteins exhibit the potential to bind to the Spike protein in different regions, as shown by a molecular docking approach. Acer, in particular, interacts with the Spike protein in the same region as in humans. Overall, we suggest that the D. melanogaster is a promising animal model for translational studies on COVID-19 associated risk factors and ACE2.     

The ACE2-binding Interface of SARS-CoV-2 Spike Inherently Deflects Immune Recognition

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID-19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.     

SARS-CoV-2 impairs the disassembly of stress granules and promotes ALS-associated amyloid aggregation

The nucleocapsid (N) protein of SARS-CoV-2 has been reported to have a high ability of liquid-liquid phase separation, which enables its incorporation into stress granules (SGs) of host cells. However, whether SG invasion by N protein occurs in the scenario of SARS-CoV-2 infection is unknow, neither do we know its consequence. Here, we used SARS-CoV-2 to infect mammalian cells and observed the incorporation of N protein into SGs, which resulted in markedly impaired self-disassembly but stimulated cell cellular clearance of SGs. NMR experiments further showed that N protein binds to the SG-related amyloid proteins via non-specific transient interactions, which not only expedites the phase transition of these proteins to aberrant amyloid aggregation in vitro, but also promotes the aggregation of FUS with ALS-associated P525L mutation in cells. In addition, we found that ACE2 is not necessary for the infection of SARS-CoV-2 to mammalian cells. Our work indicates that SARS-CoV-2 infection can impair the disassembly of host SGs and promote the aggregation of SG-related amyloid proteins, which may lead to an increased risk of neurodegeneration. Supplementary InformationThe online version contains supplementary material available at 10.1007/s13238-022-00905-7.     

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.     

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption. II. Physiological state of the host nucleoid in infected cells

Studies with ndd mutants of phage T4, deficient in the ability to induce nuclear disruption, the movement of the host DNA from a largely central location in the cell into close association with the cell membrane, show that nuclear disruption is not essential for host DNA breakdown. Degradation of prelabeled host DNA to acid-soluble products occurs at the same rate in the absence of nuclear disruption as it does in its presence. Moreover, the absence of nuclear disruption results in an alternative pathway of slow degradation of host DNA independent of phage endonuclease II.M-band analyses of association between DNA andmembrane (Earhart et al., 1968) indicate that endonuclease II is required for the release of host DNA from the membrane when nuclear disruption occurs normally, and that the product of at least one of the genes rIIA, rIIB, D1 or D2a (probably D2a, which is necessary for the synthesis of endonuclease IV) is required for DNA release when nuclear disruption does not occur.Analyses of the sizes of host DNA single strands at various times after infection by means of alkaline sucrose density-gradients show that the presence or absence of nuclear disruption has little, if any, effect on the rate of accumulation of single-strand nicks. Neutral sucrose density-gradient analyses suggest that a limited number of double-strand breaks can accumulate in host DNA when endonuclease IV is active, but few, if any, occur when neither endonuclease II or IV is active.Gentle lysis of ndd-infected cells and subsequent sedimentation analysis of the host DNA in neutral sucrose density-gradients reveal that the host chromosomes become “unfolded” within five minutes after infection. Thin-section electron microscopy shows that the host DNA becomes widely dispersed throughout the cytoplasm of cells at late times after infection with ndd mutants. These observations make it very unlikely that nuclear disruption is a passive process which occurs whenever the forces or structures which maintain the normal state of the Escherichia coli nucleoid are altered.All of our data are consistent with a mechanism of nuclear disruption which involves multiple attachment of the host DNA to the cell membrane under the control of the D2b gene of phage T4. We propose that in ndd-infected cells this multiple attachment does not occur, with the result that a limited number of double-strand breaks release much of the host DNA from the cell membrane.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.     

Generation of enzymatically competent SARS-CoV-2 decoy receptor ACE2-Fc in glycoengineered Nicotiana benthamiana

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS-CoV-2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2-fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2-Fc variant is glycosylated with mainly complex human-type N-glycans and functional with regard to enzyme activity, affinity to the SARS-CoV-2 receptor-binding domain, and wild-type virus neutralization.     

Co-expression of the SARS-CoV-2 entry molecules ACE2 and TMPRSS2 in human ovaries: Identification of cell types and trends with age

The high rate of SARS-CoV-2 infection poses a serious threat to public health. Previous studies have suggested that SARS-CoV-2 can infect human ovary, the core organ of the female reproductive system. However, it remains unclear which type of ovarian cells are easily infected by SARS-CoV-2 and whether ovarian infectivity differs from puberty to menopause. In this study, public datasets containing bulk and single-cell RNA-Seq data derived from ovarian tissues were analyzed to demonstrate the mRNA expression and protein distribution of the two key entry receptors for SARS-CoV-2—angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine protease (TMPRSS2). Furthermore, an immunohistochemical study of ACE2 and TMPRSS2 in human ovaries of different ages was conducted. Differentially expressed gene (DEG) analysis of ovaries of different ages and with varying ovarian reserves was conducted to explore the potential functions of ACE2 and TMPRSS2 in the ovary. The analysis of the public datasets indicated that the co-expression of ACE2 and TMPRSS2 was observed mostly in oocytes and partially in granulosa cells. However, no marked difference was observed in ACE2 or TMPRSS2 expression between young and old ovaries and ovaries with low and high reserves. Correspondingly, ACE2 and TMPRSS2 were detected in the human ovarian cortex and medulla, especially in oocytes of different stages, with no observed variations in their expression level in ovaries of different ages, which was consistent with the results of bioinformatic analyses. Remarkably, DEG analysis showed that a series of viral infection-related pathways were more enriched in ACE2-positive ovarian cells than in ACE2-negative ovarian cells, suggesting that SARS-CoV-2 may potentially target specific ovarian cells and affect ovarian function. However, further fundamental and clinical research is still needed to monitor the process of SARS-CoV-2 entry into ovarian cells and the long-term effects of SARS-CoV-2 infection on the ovarian function in recovered females.     

Structural basis of SARS-CoV-2 and its variants binding to intermediate horseshoe bat ACE2

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic. Intermediate horseshoe bats (Rhinolophus affinis) are hosts of RaTG13, the second most phylogenetically related viruses to SARS-CoV-2. We report the binding between intermediate horseshoe bat ACE2 (bACE2-Ra) and SARS-CoV-2 receptor-binding domain (RBD), supporting the pseudotyped SARS-CoV-2 viral infection. A 3.3 Å resolution crystal structure of the bACE2-Ra/SARS-CoV-2 RBD complex was determined. The interaction networks of Patch 1 showed differences in R34 and E35 of bACE2-Ra compared to hACE2 and big-eared horseshoe bat ACE2 (bACE2-Rm). The E35K substitution, existing in other species, significantly enhanced the binding affinity owing to its electrostatic attraction with E484 of SARS-CoV-2 RBD. Furthermore, bACE2-Ra showed extensive support for the SARS-CoV-2 variants. These results broaden our knowledge of the ACE2/RBD interaction mechanism and emphasize the importance of continued surveillance of intermediate horseshoe bats to prevent spillover risk.