Mediates Cell Entry Research Articles

The EEHV1A gH/gL complex elicits humoral and cell-mediated immune responses in mice

Elephant endotheliotropic herpesvirus (EEHV) causes lethal hemorrhagic disease (HD) in Asian and African elephants. Although rapid detection of viremia and supportive treatments may improve survival rates, an effective vaccine would mitigate the devastating effects of this virus. In elephants, chronic infection with EEHV leads to adaptive immunity against glycoproteins gB and gH/gL, the core entry machinery for most herpesviruses. We previously evaluated two EEHV gB vaccines in mice but not a gH/gL vaccine. Here, we found that inoculation of mice with an adjuvanted EEHV gH/gL subunit vaccine induced a significant antibody response that was similar to the response observed in elephants chronically infected with EEHV. Moreover, the gH/gL heterodimer elicited polyfunctional T cells with a Th1 phenotype but no detectable Th2 response. These results suggest that gH/gL, possibly in combination with gB, may be suitable immunogens for a vaccine comprising herpesvirus glycoproteins that are known to mediate cell entry and infection.

Cellular signalling by SARS-CoV-2 spike protein

Following the release of the SARS-CoV-2 genome, the spike protein was identified as the key viral protein mediating cell entry. In addition to its critical function in delivering the viral genome to the host cytoplasm, the spike protein is able to activate diverse cell signalling pathways, leading to notable cellular responses, including inflammation, cellular remodelling, and immune evasion. The spike protein is associated with the induction of a ‘cytokine storm’ characterised by elevated levels of proinflammatory cytokines like IL-6 and IL-1β. Moreover, the spike protein deregulates TGF-β and E-selectin, leading to fibrotic injury and tissue scarring in cellular remodelling, notably in pulmonary tissues. Finally, the spike protein plays a role in immune evasion, disrupting Type I interferon responses. Understanding these diverse interactions and effects is crucial for comprehending the pathogenesis of COVID-19 and developing effective therapeutic strategies.

Differentiating Cell Entry Potentials of SARS-CoV-2 Omicron Subvariants on Human Lung Epithelium Cells.

The surface spike (S) glycoprotein mediates cell entry of SARS-CoV-2 into the host through fusion at the plasma membrane or endocytosis. Omicron lineages/sublineages have acquired extensive mutations in S to gain transmissibility advantages and altered antigenicity. The fusogenicity, antigenicity, and evasion of Omicron subvariants have been extensively investigated at unprecedented speed to align with the mutation rate of S. Cells that overexpress receptors/cofactors are mostly used as hosts to amplify infection sensitivity to tested variants. However, systematic cell entry comparisons of most prior dominant Omicron subvariants using human lung epithelium cells are yet to be well-studied. Here, with human bronchial epithelium BEAS-2B cells as the host, we compared single-round virus-to-cell entry and cell-to-cell fusion of Omicron BA.1, BA.5, BQ.1.1, CH.1.1, XBB.1.5, and XBB.1.16 based upon split NanoLuc fusion readout assays and the S-pseudotyped lentivirus system. Virus-to-cell entry of tested S variants exhibited cell-type dependence. The parental Omicron BA.1 required more time to develop full entry to HEK293T-ACE2-TMPRSS2 than BEAS-2B cells. Compared to unchanged P681, S-cleavage constructs of P681H/R did not have any noticeable advantages in cell entry. Omicron BA.1 and its descendants entered BEAS-2B cells more efficiently than D614G, and it was slightly less or comparable to that of Delta. Serine protease-pretreated Omicron subvariants enhanced virus-to-cell entry in a dose-dependent manner, suggesting fusion at the plasma membrane persists as a productive cell entry route. Spike-mediated cell-to-cell fusion and total S1/S2 processing of Omicron descendants were similar. Our results indicate no obvious entry or fusion advantages of recent Omicron descendants over preceding variants since Delta, thus supporting immune evasion conferred by antigenicity shifts due to altered S sequences as probably the primary viral fitness driver.

Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.

Targeting SARS-CoV-2 and host cell receptor interactions

Despite the availability of vaccines and therapeutics, continual genetic alterations render the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) a persistent threat, particularly for the immunocompromised and elderly. Through interactions of its spike (S) protein with different receptors and coreceptors on host cell surfaces, the virus enters the cell either via fusion with the plasma membrane or through endocytosis. Angiotensin-converting enzyme 2 (ACE2) has been identified as a key receptor utilized by SARS-CoV-2 and related human coronaviruses to mediate cell entry in the lung airways. Auxiliary SARS-CoV-2 entry receptors such as ASGPR1, Kremen protein 1, integrins have also been reported. In this review, therapeutic approaches to block SARS-CoV-2 and host cell receptor interactions are discussed.

Neuropilin-1 Mediates SARS-CoV-2 Infection of Astrocytes in Brain Organoids, Inducing Inflammation Leading to Dysfunction and Death of Neurons.

Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

Systematic analysis and comparison of O-glycosylation of five recombinant spike proteins in β-coronaviruses

β-coronaviruses (β-CoVs), representative with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), depend on their highly glycosylated spike proteins to mediate cell entry and membrane fusion. Compared with the extensively identified N-glycosylation, less is known about O-glycosylation of β-CoVs S proteins, let alone its biological functions. Herein we comprehensively characterized O-glycosylation of five recombinant β-CoVs S1 subunits and revealed the macro- and micro-heterogeneity nature of site-specific O-glycosylation. We also uncovered the O-glycosylation differences between SARS-CoV-2 and its natural D614G mutant on functional domains. This work describes the systematic O-glycosylation analysis of β-CoVs S1 proteins and will help to guide the related vaccines and antiviral drugs development.

Discovery and Genomic Characterization of a Novel Henipavirus, Angavokely Virus, from Fruit Bats in Madagascar.

The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses-HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)-but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. IMPORTANCE Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described. Here, we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus that likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.

Intermediates in SARS-CoV-2 spike-mediated cell entry.

SARS-CoV-2 cell entry is completed after viral spike (S) protein–mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo–electron microscopy and cryo–electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo–electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein–directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

Integrin mediates cell entry of the SARS-CoV-2 virus independent of cellular receptor ACE2

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is broadly accepted that SARS-CoV-2 utilizes its spike protein to recognize the extracellular domain of angiotensin-converting enzyme 2 (ACE2) to enter cells for viral infection. However, other mechanisms of SARS-CoV-2 cell entry may occur. We show quantitatively that the SARS-CoV-2 spike protein also binds to the extracellular domain of broadly expressed integrin α5β1 with an affinity comparable to that of SARS-CoV-2 binding to ACE2. More importantly, we provide direct evidence that such binding promotes the internalization of SARS-CoV-2 into non-ACE2 cells in a manner critically dependent upon the activation of the integrin. Our data demonstrate an alternative pathway for the cell entry of SARS-CoV-2, suggesting that upon initial ACE2-mediated invasion of the virus in the respiratory system, which is known to trigger an immune response and secretion of cytokines to activate integrin, the integrin-mediated cell invasion of SARS-CoV-2 into the respiratory system and other organs becomes effective, thereby promoting further infection and progression of COVID-19.

Host cell entry mediators implicated in the cellular tropism of SARS‑CoV‑2, the pathophysiology of COVID‑19 and the identification of microRNAs that can modulate the expression of these mediators (Review).

The pathophysiology of coronavirus disease 2019 (COVID-19) is mainly dependent on the underlying mechanisms that mediate the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cells of the various human tissues/organs. Recent studies have indicated a higher order of complexity of the mechanisms of infectivity, given that there is a wide-repertoire of possible cell entry mediators that appear to co-localise in a cell- and tissue-specific manner. The present study provides an over-view of the 'canonical' SARS-CoV-2 mediators, namely angiotensin converting enzyme 2, transmembrane protease serine 2 and 4, and neuropilin-1, expanding on the involvement of novel candidates, including glucose-regulated protein 78, basigin, kidney injury molecule-1, metabotropic glutamate receptor subtype 2, ADAM metallopeptidase domain 17 (also termed tumour necrosis factor-α convertase) and Toll-like receptor 4. Furthermore, emerging data indicate that changes in microRNA (miRNA/miR) expression levels in patients with COVID-19 are suggestive of further complexity in the regulation of these viral mediators. An in silico analysis revealed 160 candidate miRNAs with potential strong binding capacity in the aforementioned genes. Future studies should concentrate on elucidating the association between the cellular tropism of the SARS-CoV-2 cell entry mediators and the mechanisms through which they might affect the clinical outcome. Finally, the clinical utility as a biomarker or therapeutic target of miRNAs in the context of COVID-19 warrants further investigation.

Epidermal growth factor receptors siRNA-conjugated collagen modified gold nanoparticles for targeted imaging and therapy of lung cancer

Epidermal growth factor receptor (EGFR), a critical factor promotes lung cancer cell proliferation and survival. Knockdown of EGFR expression thus promise beating lung cancer clinically. Functionalized gold nanoparticles may serve an effective vehicle carrying theranostic bio-active materials. Herein, physically gold nanoparticles were fabricated with biocompatible collagen to improve siRNA loading capacity carrying EGFR siRNA to treat lung cancer. Physic-chemical properties were comprehensively characterized for the collagen gold nanoparticle (C–Au), and with EGFR siRNA conjugation, C–Au-EGFRsi namely. Issues of biocompatibility were addressed. Interestingly, C–Au appeared more biocompatible to normal airway epithelial cells (BEAS-2B) than to cancer cells (A549) in terms of ROS production, cell cycle behavior, and cell growth influence. The C–Au demonstrated comparable or even more efficient, compared with lipofetamine, in carrying siRNA to knockdown EGFR of A549 cells. Endocytosis mediated cell entry for the collagen gold nanoparticles, and endosome-lysosomal pathway involved transporting and metabolizing these nanoparticles. In xenograft mice model, substantial tumor suppression effects were observed treating with C–Au-EGFRsi, in which tumor weight reduced 30% for lipofetamine carrier, and down to 70% for C–Au carrier. Particularly, overall survival rate was improved for both treatment groups with lipofetamine and C–Au carrier, respectively.

Prediction of Non-canonical Routes for SARS-CoV-2 Infection in Human Placenta Cells.

The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the mechanisms associated with intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non- canonical mediators of SARS-CoV-2 infection and replication in the placenta. Despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2, we show that cells of the syncytiotrophoblast, villous cytotrophoblast, and extravillous trophoblast co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL. We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.

Computational insights into differential interaction of mammalian angiotensin-converting enzyme 2 with the SARS-CoV-2 spike receptor binding domain

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Angiotensin-converting enzyme 2 (ACE2) has been identified as the host cell receptor that binds to the receptor-binding domain (RBD) of the SARS-COV-2 spike protein and mediates cell entry. Because the ACE2 proteins are widely available in mammals, it is important to investigate the interactions between the RBD and the ACE2 of other mammals. Here we analyzed the sequences of ACE2 proteins from 16 mammals, predicted the structures of ACE2-RBD complexes by homology modeling, and refined the complexes using molecular dynamics simulation. Analyses on sequence, structure, and dynamics synergistically provide valuable insights into the interactions between ACE2 and RBD. The analysis outcomes suggest that the ACE2 of bovine, cat, and panda form strong binding interactions with RBD, while in the cases of rat, least horseshoe bat, horse, pig, mouse, and civet, the ACE2 proteins interact weakly with RBD.

Integrin activation is an essential component of SARS-CoV-2 infection

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's β subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

Hypoxia alters the expression of ACE2 and TMPRSS2 SARS-CoV-2 cell entry mediators in hCMEC/D3 brain endothelial cells.

The mechanisms by which the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces neurological complications remain to be elucidated. We aimed to identify possible effects of hypoxia on the expression of SARS-CoV-2 cell entry mediators, angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) protein, in human brain endothelial cells, in vitro. hCMEC/D3 cells were exposed to different oxygen tensions: 20% (Control group), 8% or 2% O2 (Hypoxia groups). Cells were harvested 6-, 24- and 48 h following hypoxic challenge for assessment of mRNA and protein, using qPCR and Western Blot. The response of the brain endothelial cells to hypoxia was replicated using modular incubator chambers. We observed an acute increase (6 h, p < 0.05), followed by a longer-term decrease (48 h, p < 0.05) in ACE2 mRNA and protein expression, accompanied by reduced expression of TMPRSS2 protein levels (48 h, p < 0.05) under the more severe hypoxic condition (2% O2). No changes in levels of von Willebrand Factor (vWF – an endothelial cell damage marker) or interleukin 6 (IL-6 – a pro-inflammatory cytokine) mRNA were observed. We conclude that hypoxia regulates brain endothelial cell ACE2 and TMPRSS2 expression in vitro, which may indicate human brain endothelial susceptibility to SARS-CoV-2 infection and subsequent brain sequelae.

The intersection of genetics and COVID-19 in 2021: preview of the 2021 Rodney Howell Symposium

The intersection of genetics and COVID-19 in 2021: preview of the 2021 Rodney Howell Symposium

Viral Interactions with Adaptor-Protein Complexes: A Ubiquitous Trait among Viral Species.

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.

ACE2, TMPRSS2, and L-SIGN Expression in Placentae From HIV-Positive Pregnancies Exposed to Antiretroviral Therapy-Implications for SARS-CoV-2 Placental Infection.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding receptor ACE2 and the spike protein priming protease TMPRSS2 are coexpressed in human placentae. It is unknown whether their expression is altered in the context of HIV infection and antiretroviral therapy (ART).MethodsWe compared mRNA levels of SARS-CoV-2 cell-entry mediators ACE2, TMPRSS2, and L-SIGN by quantitative polymerase chain reaction in 105 placentae: 45 from pregnant women with HIV (WHIV) on protease inhibitor (PI)-based ART, 17 from WHIV on non-PI–based ART, and 43 from HIV-uninfected women.ResultsACE2 levels were lower, while L-SIGN levels were higher, in placentae from WHIV on PI-based ART compared to those on non-PI–based ART and to HIV-uninfected women. TMPRSS2 levels were similar between groups. Black race was significantly associated with lower expression of ACE2 and higher expression of L-SIGN. ACE2 levels were significantly higher in placentae of female fetuses.ConclusionsWe identified pregnant women of black race and WHIV on PI-based ART to have relatively lower expression of placental ACE2 than those of white race and HIV-uninfected women. This may potentially contribute to altered susceptibility to COVID-19 in these women, favorably by reduced viral entry or detrimentally by loss of ACE2 protection against hyperinflammation.

COVID‑19 and SARS‑CoV‑2 host cell entry mediators: Expression profiling of TMRSS4 in health and disease.

Severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2), the causative viral agent for the ongoing COVID-19 pandemic, enters its host cells primarily via the binding of the SARS-CoV-2 spike (S) proteins to the angiotensin-converting enzyme 2 (ACE2). A number of other cell entry mediators have also been identified, including neuropilin-1 (NRP1) and transmembrane protease serine 2 (TMPRSS2). More recently, it has been demonstrated that transmembrane protease serine 4 (TMPRSS4) along with TMPRSS2 activate the SARS-CoV-2 S proteins, and enhance the viral infection of human small intestinal enterocytes. To date, a systematic analysis of TMPRSS4 in health and disease is lacking. In the present study, using in silico tools, the gene expression and genetic alteration of TMPRSS4 were analysed across numerous tumours and compared to controls. The observations were also expanded to the level of the central nervous system (CNS). The findings revealed that TMPRSS4 was overexpressed in 11 types of cancer, including lung adenocarcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, thyroid carcinoma, ovarian cancer, cancer of the rectum, pancreatic cancer, colon and stomach adenocarcinoma, uterine carcinosarcoma and uterine corpus endometrial carcinoma, whilst it was significantly downregulated in kidney carcinomas, acute myeloid leukaemia, skin cutaneous melanoma and testicular germ cell tumours. Finally, a high TMPRSS4 expression was documented in the olfactory tubercle, paraolfactory gyrus and frontal operculum, all brain regions which are associated with the sense of smell and taste. Collectively, these data suggest that TMPRSS4 may play a role in COVID-19 symptomatology as another SARS-CoV-2 host cell entry mediator responsible for the tropism of this coronavirus both in the periphery and the CNS.