ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes - evidence from a translational clinical and experimental approach.

Abstract

SARS-CoV-2 infects host cells via an ACE2/TMPRSS2 entry mechanism. Monocytes and macrophages, which play a key role during severe COVID-19 express only low or no ACE2, suggesting alternative entry mechanisms in these cells. In silico analyses predicted GRP78, which is constitutively expressed on monocytes and macrophages, to be a potential candidate receptor for SARS-CoV-2 virus entry. Hospitalized COVID-19 patients were characterized regarding their pro-inflammatory state and cell surface GRP78 (csGRP78) expression in comparison to healthy controls. RNA from CD14⁺ monocytes of patients and controls were subjected to transcriptome analysis that was specifically complemented by bioinformatic re-analyses of bronchoalveolar lavage fluid (BALF) datasets of COVID-19 patients with a focus on monocyte/macrophage subsets, SARS-CoV-2 infection state as well as GRP78 gene expression. Monocyte and macrophage immunohistocytochemistry on GRP78 was conducted in post-mortem lung tissues. SARS-CoV-2 spike and GRP78 protein interaction was analyzed by surface plasmon resonance, GST Pull-down and Co-Immunoprecipitation. SARS-CoV-2 pseudovirus or single spike protein uptake was quantified in csGRP78^high THP-1 cells. Cytokine patterns, monocyte activation markers and transcriptomic changes indicated typical COVID-19 associated inflammation accompanied by upregulated csGRP78 expression on peripheral blood and lung monocytes/macrophages. Subsequent cell culture experiments confirmed an association between elevated pro-inflammatory cytokine levels and upregulation of csGRP78. Interaction of csGRP78 and SARS-CoV-2 spike protein with a dissociation constant of K_D = 55.2 nM was validated in vitro. Infection rate analyses in ACE2^low and GRP78^high THP-1 cells showed increased uptake of pseudovirus expressing SARS-CoV-2 spike protein. Our results demonstrate that csGRP78 acts as a receptor for SARS-CoV-2 spike protein to mediate ACE2-independent virus entry into monocytes. Funded by the Sino-German-Center for Science Promotion (C-0040) and the Germany Ministry BMWi/K [DLR-grant 50WB1931 and RP1920 to AC, DM, TW].