Bedeutung der ACE2-Spaltung durch Wirtszellproteasen für die SARS-Coronavirus-Infektion

Abstract

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a highly pathogenic virus and its zoonotic entry into the human population represents a substantial health threat. The identification of host cell factors important for SARS-CoV spread and pathogenesis might yield new targets for therapy. The SARS-CoV envelope protein spike (S) binds to the cellular receptor, Angiotensin Converting Enzyme 2 (ACE2), and mediates viral entry into target cells. Cleavage and activation of the S protein by host cell proteases is essential for the infectious S protein-mediated entry. The type II transmembrane serine proteases (TTSPs) TMPRSS2 and HAT cleave and activate the S protein, at least upon expression in cell culture. Whether these enzymes are expressed in human lung cells, the target cells of SARS-CoV infection, was unclear and was to be investigated in the present thesis. TMPRSS2 and HAT also cleave the viral receptor ACE2 and it was postulated that ACE2 cleavage increases viral entry into host cells. However, the underlying mechanism was not known and was to be analyzed within the present study. It could be shown that TMPRSS2, HAT and ACE2 are coexpressed in epithelial cells of the respiratory tract. Therefore, these proteases could promote the spread of SARS-CoV in lung epithelium. Furthermore, an amino acid sequence in ACE2, which is essential for the processing by TMPRSS2 and HAT, was identified. The functional analysis of ACE2 mutants demonstrated that cleavage at this site increases S protein-driven host cell entry, and immunofluorescence studies provided evidence that the augmented entry efficiency was due to increased viral particle uptake into the cell. Finally, it was demonstrated that TMPRSS2 and another cellular protease, A Disintegrin And Metalloproteinase 17 (ADAM17), compete for ACE2 cleavage and the cleavage site for ADAM17 in ACE2 could be identified. However, ACE2 cleavage by ADAM17 was found to be dispensable for the S protein-driven cell entry. In summary, these studies indicate that TMPRSS2 and HAT promote SARS-CoV infection by cleavage of the viral S protein and its receptor. The proteases therefore constitute potential targets for antiviral intervention.