In silico mutagenesis of human ACE2 with S protein and translational efficiency explain SARS-CoV-2 infectivity in different species

The coronavirus disease COVID-19 constitutes the most severe pandemic of the last decades having caused more than 1 million deaths worldwide. The SARS-CoV-2 virus recognizes the angiotensin converting enzyme 2 (ACE2) on the surface of human cells through its spike protein. It has been reported that the coronavirus can mildly infect cats, and ferrets, and perhaps dogs while not pigs, mice, chicken and ducks. Differences in viral infectivity among different species or individuals could be due to amino acid differences at key positions of the host proteins that interact with the virus, the immune response, expression levels of host proteins and translation efficiency of the viral proteins among other factors. Here, first we have addressed the importance that sequence variants of different animal species, human individuals and virus isolates have on the interaction between the RBD domain of the SARS-CoV-2 spike S protein and human angiotensin converting enzyme 2 (ACE2). Second, we have looked at viral translation efficiency by using the tRNA adaptation index. We find that integration of both interaction energy with ACE2 and translational efficiency explains animal infectivity. Humans are the top species in which SARS-CoV-2 is both efficiently translated as well as optimally interacting with ACE2. We have found some viral mutations that increase affinity for hACE and some hACE2 variants affecting ACE2 stability and virus binding. These variants suggest that different sensitivities to coronavirus infection in humans could arise in some cases from allelic variability affecting ACE2 stability and virus binding.     

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.     

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Evolutionary pathways to SARS-CoV-2 resistance are opened and closed by epistasis acting on ACE2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

This study suggests that ancient events in mammalian cardiovascular evolution determined the host range of SARS-CoV-2 millions of years before the current pandemic. These physiological constraints are so inflexible that escape from SARS-CoV-2 susceptibility would likely have required significant alterations to the human cardiovascular system.     

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.     

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.     

Insights on cross-species transmission of SARS-CoV-2 from structural modeling

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 31 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic and confirmed cases of cross-species transmission, the question of the extent to which this transmission is possible emerges, as well as what molecular features distinguish susceptible from non-susceptible animal species. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also allow us to predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.     

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.     

TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-γ (IFN-γ)- induced ACE2 expression through a mechanism involving upregulating IFN-γ receptor 2 (IFNGR2) in both A549 and PAEpiCs. The upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.     

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.     

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury(AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells(KPTECs) in 2 D and 3 D culture systems using conditional reprogramming(CR) and organoids techniques.These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters(SLC34 A3 and cubilin). They also expressed angiotensin-converting enzyme 2(ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34 A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3 D organoids culture compared to that in 2 D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike(S) protein was able to enter CR cells with luciferase reporter. This integrated 2 D CR and 3 D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.     

Single-cell transcriptome analysis of the novel coronavirus (SARS-CoV-2) associated gene ACE2 expression in normal and non-obstructive azoospermia (NOA) human male testes

Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2(ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq(scRNA-seq) analysis on 853 male embryo primordial germ cells(PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia(NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors' Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.     

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.     

Single-cell RNA analysis on ACE2 expression provides insights into SARS-CoV-2 potential entry into the bloodstream and heart injury

Coronavirus disease-2019 (COVID-19) is a global pandemic with high infectivity and pathogenicity, accounting for tens of thousands of deaths worldwide. Recent studies have found that the pathogen of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), shares the same cell receptor angiotensin converting enzyme II (ACE2) as SARS-CoV. The pathological investigation of COVID-19 deaths showed that the lungs had characteristics of pulmonary fibrosis. However, how SARS-CoV-2 spreads from the lungs to other organs has not yet been determined. Here, we performed an unbiased evaluation of cell-type-specific expression of ACE2 in healthy and fibrotic lungs, as well as in normal and failed adult human hearts, using published single-cell RNA-seq data. We found that ACE2 expression in fibrotic lungs mainly locates in arterial vascular cells, which might provide a route for bloodstream spreading of SARS-CoV-2. Failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, and SARS-CoV-2 might attack cardiomyocytes through the bloodstream in patients with heart failure. Moreover, ACE2 was highly expressed in cells infected by respiratory syncytial virus or Middle East respiratory syndrome coronavirus and in mice treated by lipopolysaccharide. Our findings indicate that patients with pulmonary fibrosis, heart failure, and virus infection have a higher risk and are more susceptible to SARS-CoV-2 infection. The SARS-CoV-2 might attack other organs by getting into the bloodstream. This study provides new insights into SARS-CoV-2 blood entry and heart injury and might propose a therapeutic strategy to prevent patients from developing severe complications.     

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.     

ACE2 coding variants in different populations and their potential impact on SARS-CoV-2 binding affinity

The susceptibility of different populations to SARS-CoV-2 infection is not yet understood. Here, we combined ACE2 coding variants' analysis in different populations and computational chemistry calculations to probe the effects on SARS-CoV-2/ACE2 interaction. ACE2-K26R; which is most frequent in Ashkenazi Jewish population decreased the SARS-CoV-2/ACE2 electrostatic attraction. On the contrary, ACE2-I468V, R219C, K341R, D206G, G211R increased the electrostatic attraction; ordered by binding strength from weakest to strongest. The aforementioned variants are most frequent in East Asian, South Asian, African and African American, European, European and South Asian populations, respectively.     

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.     

ACE2 Correlated With Immune Infiltration Serves As A Novel Prognostic Biomarker In Clear Cell Renal Cell Carcinoma: Implication For COVID-19

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global infection, and is seriously threatening human life, especially cancer patients. Thus, we sought to determine the clinical roles of ACE2 (the cell entry receptor of SARS-CoV-2) in ccRCC (clear cell renal cell carcinoma). TCGA, GEO and TIP datasets, and immunohistochemistry and western blot results were used to determine the prognostic and clinicopathological characteristics of ACE2. ACE2 expression was down-regulated in ccRCC tissues and cell lines. The multivariate Cox regression analysis results indicated that increased ACE2 expression was independent predictor of longer OS (HR: 0.8259, 95%CI: 0.7734-0.8819, P<0.0001) and RFS (HR: 0.8023, 95%CI: 0.7375-0.8729, P<0.0001) in ccRCC patients. Lower ACE2 expression was also associated with advanced tumor stage, higher histological grade and pathological stage, and metastasis. Besides, ACE2 expression was significantly positively and negatively correlated with CD4 Naïve infiltration and CD4 Memory infiltration, respectively. Moreover, higher CD4 Naïve and lower CD4 Memory infiltration levels were associated with better pathological features and longer OS and RFS. Furthermore, high ACE2 expression group in decreased CD4 Naïve, enriched CD4 Naïve and enriched CD4 memory cohort had favorable prognosis. These findings identified that AEC2 was significantly reduced in ccRCC, and decreased ACE2 was related to worse pathological features and poor prognosis. Low ACE2 expression in ccRCC may partially affect the prognosis due to altered immune cells infiltration levels.     

Analysis of SARS-CoV-2 infection associated cell entry proteins ACE2, CD147, PPIA,and PPIB in datasets from non SARS-CoV-2 infected neuroblastoma patients,as potential prognostic and infection biomarkers in neuroblastoma

SARS-CoV-2 viral contagion has given rise to a worldwide pandemic. Although most children experience minor symptoms from SARS-CoV-2 infection, some have severe complications including Multisystem Inflammatory Syndrome in Children. Neuroblastoma patients may be at higher risk of severe infection as treatment requires immunocompromising chemotherapy and SARS-CoV-2 has demonstrated tropism for nervous cells. To date, there is no sufficient epidemiological data on neuroblastoma patients with SARS-CoV-2. Therefore, we evaluated datasets of non-SARS-CoV-2 infected neuroblastoma patients to assess for key genes involved with SARS-CoV-2 infection as possible neuroblastoma prognostic and infection biomarkers. We hypothesized that ACE2, CD147, PPIA and PPIB, which are associated with viral-cell entry, are potential biomarkers for poor prognosis neuroblastoma and SARS-CoV-2 infection.We have analysed three publicly available neuroblastoma gene expression datasets to understand the specific molecular susceptibilities that high-risk neuroblastoma patients have to the virus. Gene Expression Omnibus (GEO) GSE49711 and GEO GSE62564 are the microarray and RNA-Seq data, respectively, from 498 neuroblastoma samples published as part of the Sequencing Quality Control initiative. TARGET, contains microarray data from 249 samples and is part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. ACE2, CD147, PPIA and PPIB were identified through their involvement in both SARS-CoV-2 infection and cancer pathogenesis.In-depth statistical analysis using Kaplan-Meier, differential gene expression, and Cox multivariate regression analysis, demonstrated that overexpression of ACE2, CD147, PPIA and PPIB is significantly associated with poor-prognosis neuroblastoma samples. These results were seen in the presence of amplified MYCN, unfavourable tumour histology and in patients older than 18 months of age. Previously, we have shown that high levels of the nerve growth factor receptor NTRK1 together with low levels of the phosphatase PTPN6 and TP53 are associated with increased relapse-free survival of neuroblastoma patients. Interestingly, low levels of expression of ACE2, CD147, PPIA and PPIB are associated with this NTRK1-PTPN6-TP53 module, suggesting that low expression levels of these genes are associated with good prognosis. These findings have implications for clinical care and therapeutic treatment. The upregulation of ACE2, CD147, PPIA and PPIB in poor-prognosis neuroblastoma samples suggests that these patients may be at higher risk of severe SARS-CoV-2 infection. Importantly, our findings reveal ACE2, CD147, PPIA and PPIB as potential biomarkers and therapeutic targets for neuroblastoma.