The novel coronavirus sars-cov-2 binds rgd Integrins and upregulates avb3 integrins in Covid-19 infected lungs

ABSTRACT

The novel coronavirus SARS-CoV-2 utilizes Angiotensin Converting Enzyme-2 (ACE2) receptors to internalize cells, which are expressed in the nasal and ocular mucosa, and at low levels in the pulmonary epithelium. Despite significant sequence similarities there are substantial differences in transmission dynamics and clinical phenotype between SARS-CoV-2 and SARS-CoV-1. The SARS-CoV-2 spike protein (S1), which is used to internalize cells, contains RGD integrin binding domains which are not present within SARS-CoV-1 S1. We investigated whether SARS-CoV-2 S1 binds integrins while exploring mechanisms that might upregulate ACE2 expression to help explain SARS-CoV-2 viral entry and associated respiratory disease. Lung cell line ACE2 expression was determined using QPCR and western blotting, and in primary lung cells using single cell RNAseq data from publicly available datasets. The effect of IL6 and TGFb on ACE2 expression levels in lung epithelial cells and precision cut lung slices (PCLS) was explored. Solid phase binding assays were used to investigate S1 binding to ACE2 or av containing integrins. Immunohistochemistry was used to stain sections of COVID-19 infected lung tissue for ACE2 and av containing integrins. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 and only a small proportion of Alveolar type 2 epithelial cells are ACE2 positive (1.5%). Supporting this we found low level ACE2 mRNA and protein expression in small airway epithelial cells, immortalized human bronchial epithelial cells (iHBECs) and A549 cells. IL6 had no effect on ACE2 mRNA or protein expression in the above cells, nor did it affect ACE2 protein in PCLS. TGFb increased ACE2 mRNA in iHBECs and increased ACE2 protein in PCLS. Binding assays demonstrated that SARS-CoV-2 S1 binds avb3 and avb6 integrins in an RGD dependent manner, albeit with a lower affinity than to ACE2. Crucially avb3 integrins are upregulated in COVID-19 infected lung tissue, whereas ACE2 levels remain low even in patients with high viral RNA and protein expression in alveolar tissue. Our data suggests SARS-CoV-2 is able to bind integrins, and may utlise this mechanism to facilitate internalization into lung epithelial cells, which may help explain severe pathology despite low ACE2 expression levels in the lung.