heparitin-sulfate and Coronavirus-Infections

Pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 19 disease (COVID-19) which presents a large spectrum of manifestations with fatal outcomes in vulnerable people over 70-years-old and with hypertension, diabetes, obesity, cardiovascular disease, COPD, and smoking status. Knowledge of the entry receptor is key to understand SARS-CoV-2 tropism, transmission and pathogenesis. Early evidence pointed to angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-2 entry receptor. Here, we provide a critical summary of the current knowledge highlighting the limitations and remaining gaps that need to be addressed to fully characterize ACE2 function in SARS-CoV-2 infection and associated pathogenesis. We also discuss ACE2 expression and potential role in the context of comorbidities associated with poor COVID-19 outcomes. Finally, we discuss the potential co-receptors/attachment factors such as neuropilins, heparan sulfate and sialic acids and the putative alternative receptors, such as CD147 and GRP78.

Topics: Angiotensin-Converting Enzyme 2; Basigin; Betacoronavirus; Comorbidity; Coronavirus Infections; COVID-19; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Enzymologic; Heparitin Sulfate; Humans; Hypertension; Neuropilin-1; Oligopeptides; Organ Specificity; Pandemics; Peptidyl-Dipeptidase A; Pneumonia, Viral; Protein Binding; Receptors, Virus; Renin-Angiotensin System; Respiratory System; RNA, Messenger; SARS-CoV-2; Sialic Acids; Spike Glycoprotein, Coronavirus; Virus Attachment; Virus Internalization

We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.

Topics: Amino Acid Sequence; Angiotensin-Converting Enzyme 2; Betacoronavirus; Binding Sites; Cell Line; Coronavirus Infections; COVID-19; Heparin; Heparitin Sulfate; Humans; Kidney; Lung; Molecular Dynamics Simulation; Pandemics; Peptidyl-Dipeptidase A; Pneumonia, Viral; Protein Binding; Protein Domains; Recombinant Proteins; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

Topics: Aged; Betacoronavirus; Coronavirus Infections; COVID-19; Creatinine; Critical Care; Cross-Sectional Studies; Endothelium; Female; Glucuronidase; Heparin Antagonists; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Humans; Interleukin-6; L-Lactate Dehydrogenase; Male; Middle Aged; Pandemics; Pneumonia, Viral; SARS-CoV-2; Tight Junctions

It is well known that many viruses use heparan sulfate as the initial attachment factor. In the present study, we determined whether porcine epidemic diarrhea virus (PEDV), an emerging veterinary virus, infects Vero cells by attaching to heparan sulfate. Western blot analysis, real-time PCR, and plaque formation assay revealed that PEDV infection was inhibited when the virus was pretreated with heparin (an analogue of heparan sulfate). There was no inhibitory effect when the cells were pre-incubated with heparin. We next demonstrated that enzymatic removal of the highly sulfated domain of heparan sulfate by heparinase I treatment inhibited PEDV infection. We also confirmed that sodium chlorate, which interferes with heparan sulfate biosynthesis, also inhibited PEDV infection. Furthermore, we examined the effect of two heparin derivatives with different types of sulfation on PEDV infection. The data suggested de-N-sulfated heparin, but not N-acetyl-de-O-sulfated heparin, inhibits PEDV infection. In summary, our studies revealed that heparan sulfate acts as the attachment factor of PEDV in Vero cells.

Topics: Animals; Chlorocebus aethiops; Coronavirus Infections; Heparitin Sulfate; Porcine epidemic diarrhea virus; Receptors, Virus; Swine; Swine Diseases; Vero Cells; Virus Attachment

Bioluminescence imaging (BLI) is a powerful new method to study virus dissemination in the live animal. Here we used this method to monitor the spatial and temporal progression of mouse hepatitis coronavirus (MHV) infection in mice using luciferase-expressing viruses. Upon intranasal inoculation, virus replication could initially be observed in the nasal cavity and the cervical lymph nodes, after which the infection spread to the brain and frequently to the eyes. The kinetics of virus spread to and clearance from the brain appeared to depend on the inoculation dose. After intraperitoneal inoculation, virus replication was predominantly observed in the liver and occasionally in the intestines, but interestingly also in the tail and paws. BLI thus elucidated new anatomic locations of virus replication. Furthermore, MHV dissemination was shown to be critically depended on the viral spike protein, but also on the mouse strain used. Widespread dissemination was observed in mice lacking a functional type I interferon response. The importance of the type I interferon system in limiting viral spread was also demonstrated by the administration of type I interferons to mice. Our results provide new insights in coronavirus pathogenesis and demonstrate the potential of BLI to study coronavirus-host interactions in vivo.

Topics: Animals; Coronavirus Infections; Diagnostic Imaging; Heparitin Sulfate; Interferon Type I; Luminescent Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Murine hepatitis virus; Recombinant Proteins; Virus Replication

A rapid antiviral immune response may be related to viral interaction with the host cell leading to activation of macrophages via pattern recognition receptors (PPRs) or specific viral receptors. Carcinoembryonic cell adhesion antigen 1a (CEACAM1a) is the specific receptor for the mouse hepatitis virus (MHV), a coronavirus known to induce acute viral hepatitis in mice. The objective of this study was to understand the mechanisms responsible for the secretion of high-pathogenic MHV3-induced inflammatory cytokines. We report that the induction of the pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in peritoneal macrophages does not depend on CEACAM1a, as demonstrated in cells isolated from Ceacam1a(-/-) mice. The induction of IL-6 and TNF-alpha production was related rather to the fixation of the spike (S) protein of MHV3 on Toll-like receptor 2 (TLR2) in regions enriched in heparan sulphate and did not rely on viral replication, as demonstrated with denatured S protein and UV-inactivated virus. High levels of IL-6 and TNF-alpha were produced in livers from infected C57BL/6 mice but not in livers from Tlr2(-/-) mice. The histopathological observations were correlated with the levels of those inflammatory cytokines. Depending on mouse strain, the viral fixation to heparan sulfate/TLR2 stimulated differently the p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB in the induction of IL-6 and TNF-alpha. These results suggest that TLR2 and heparan sulphate receptors can act as new viral PPRs involved in inflammatory responses.

Topics: Animals; Butadienes; Carcinoembryonic Antigen; Cell Line; Coronavirus Infections; Enzyme Inhibitors; Fibroblasts; Heparitin Sulfate; Hepatitis, Animal; Imidazoles; Interleukin-6; Liver; Macrophages, Peritoneal; Membrane Glycoproteins; Mice; Mice, Knockout; Murine hepatitis virus; NF-kappaB-Inducing Kinase; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Spike Glycoprotein, Coronavirus; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha; Viral Envelope Proteins; Virus Replication

A highly neurovirulent mouse hepatitis virus (MHV) JHMV strain (wt) with receptor (MHVR)-independent infection activity and its low-virulent mutant srr7 without such activity were found to attach to MHVR-negative, non-permissive BHK cells. To identify the molecule that interacts with JHMV, we focused on heparan sulfate (HS) since it works as a receptor of a mutant MHV-rec1 that infects in an MHVR-independent fashion. The present study indicates that HS interacts with both wt JHMV and srr7 but it does not function as an entry receptor as it apparently does for MHV-rec1. Furthermore, HS failed to serve as an entry receptor in the MHVR-independent infection of wt JHMV, indicating that HS is not a host factor that wt JHMV utilizes in an MHVR-independent infection.

Topics: Amino Acid Sequence; Animals; Binding Sites; Carcinoembryonic Antigen; Cell Line; Cell Membrane; Coronavirus; Coronavirus Infections; Cricetinae; Heparitin Sulfate; Kidney; Mice; Protein Binding; Receptors, Virus; Virulence