Cell surface GRP78 functions as an alternative virus entry receptor on monocytes during SARS-CoV- 2 infection

Background: It has been widely acknowledged that severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) infects host cells via the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) entry mechanism. However, ACE2 and TMPRSS2 cannot explain the Toll-like receptor driven response of monocytes since there is no ACE2 expressed on monocytes, suggesting alternative receptor(s) on these cells. Here, we report cell surface glucose-regulated protein 78 (csGRP78) which is abundantly expressed on monocytes to function as an alternative receptor for SARS-CoV- 2 internalization. Method(s): Blood from COVID-19 patients and healthy donors were collected for csGRP78 and monocyte activation marker as well as cytokine concentration. In vitro SPR, GST pull-down and Co-IP assay were used to determine interaction between SARS-CoV- 2 spike protein and GRP78. Cytokine mixture of IL-1beta, IL-6, TNF and IFN-gamma were used to stimulated csGRP78 upregulation on human monocytic cell line THP-1. GRP78-overexpressing- THP- 1 was also established. pseudo-typed virus expressing spike protein was used to infect mock or GRP78 over-expressing THP-1 cells. Result(s): Our results show that csGRP78 is upregulated on the monocyte of COVID-19 patients. Moreover, in vitro cell culture experiments revealed that stimulation of wtTHP-1 and GRP78 over-expressing THP-1 with the relevant cytokines IL-1beta, IL-6, TNF and IFN-gamma induces similar csGRP78 and activation marker upregulation on cell surface as found on patients' monocytes. In vitro spike protein and GRP78 interaction tests, confirmed direct binding of spike protein and GRP78. Finally, pseudo-typed virus infection assay showed that virus entered GRP78 over-expressing THP-1 cells but not control THP-1 cells. Conclusion(s): Our results demonstrate that csGRP78 acts as a potential functional receptor for SARS-CoV- 2 spike protein and mediates ACE2 independent SARS-CoV- 2 entry into monocytes. These findings provide insight into role of monocytes in the pathophysiology of COVID-19, and suggest a new therapeutic target candidate for anti-SARS- CoV- 2 treatment.

Cell surface GRP78 functions as an alternative virus entry receptor on monocytes during SARS-CoV-2 infection

It has been widely acknowledged that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) entry mechanism. However, ACE2 and TMPRSS2 cannot explain the Toll-like receptor driven response of monocytes since there is no ACE2 expressed on monocytes, suggesting alternative receptor(s) on these cells. Here, we report cell surface glucose-regulated protein 78 (csGRP78) which is abundantly expressed on monocytes to function as an alternative receptor for SARS-CoV-2 internalization. Our results show that csGRP78 is upregulated on the monocyte of COVID-19 patients. Moreover, in vitro cell culture experiments revealed that GRP78 over-expressing THP-1 cells and stimulation of wtTHP-1 cells with the relevant cytokines IL-1beta, IL-6, TNF and IFN-gamma induces similar csGRP78 and activation marker upregulation on cell surface as found on patients' monocytes. In vitro spike protein and GRP78 interaction tests (SPR assay, GST-pull down and Co-IP), confirmed direct binding of spike protein and GRP78. Finally, pseudo-typed virus expressing spike protein was used to infect mock or GRP78 over-expressing THP-1 cells. We found that pseudo-typed virus entered GRP78 over-expressing THP-1 cells but not control THP-1 cells. Our results demonstrate that csGRP78 acts as a potential functional receptor for SARS-CoV-2 spike protein and mediates ACE2 independent SARS-CoV-2 entry into monocytes. These findings provide insight into role of monocytes in the pathophysiology of COVID-19, and suggest a new therapeutic target candidate for anti-SARS-CoV2 treatment.