TMPRSS2 and ADAM17 Cleave ACE2 Differentially and Only Proteolysis by TMPRSS2 Augments Entry Driven by the Severe Acute Respiratory Syndrome Coronavirus Spike Protein

Abstract

bThe type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors. C oronaviruses are enveloped RNA viruses which cause enteric, respiratory, and central nervous system diseases in a variety of animals and humans (1). The coronaviruses NL63, 229E, and OC43 are adapted to spread in humans, and infection is usually associated with mild respiratory symptoms (2-8). In contrast, the zoonotic transmission of animal coronaviruses to humans can result in novel, severe diseases. The severe acute respiratory syndrome coronavirus (SARS-CoV), which is believed to have been transmitted from bats via an intermediate host to humans (9-11), is the causative agent of the respiratory disease SARS, which claimed more than 700 lives in 2002-2003 (12). Similarly, the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) induces a severe, SARS-related respiratory disease, and its spread is at present responsible for 64 deaths (13,14). The elucidation of the molecular processes underlying the spread and pathogenesis of highly pathogenic coronaviruses is required to devise effective antiviral strategies and is therefore the focus of current research efforts.The coronavirus surface protein spike (S) mediates entry into target cells by binding to a cellular receptor and by subsequently fusing the viral envelope with a host cell membrane (15, 16). The receptor bin...

Supramolecular Architecture of the Coronavirus Particle

“…One or more protease cleavage events are necessary to prime S for membrane fusion. An apparent fusion peptide of coronaviruses resides in the S2 region (Bosch et al, 2003;Sainz et al, 2005), where it is exposed by cleavage that takes place on tetraspanin-enriched membranes where host proteases including TMPRSS2 and HAT localize for some coronaviruses including MERS-CoV, while other spikes can be primed for entry by cathepsins (Bertram et al, 2013;Earnest et al, 2015;Glowacka et al, 2011;Heurich et al, 2014;Huang et al, 2006;Shulla et al, 2011;Simmons et al, 2005). This is consistent with host gene knockdown experiments that show that coronavirus entry is dependent on several elements that are important in the endosomal and lysosomal trafficking (Burkard et al, 2014;Wong et al, 2015).…”

Structure of dimeric full-length human ACE2 in complex with B0AT1

“…For membrane fusion, cleavage of the C-terminal segment, especially residues 697 to 716 ( Supplementary Figure 4), of ACE2 by proteases, such as transmembrane protease serine 2 (TMPRSS2), can enhance S protein-driven viral entry (33,34). In our present structure, residues of 697-716 form helix N3 and N4 in the Neck domain and map to the dimeric interface of ACE2.…”

Bat-to-human: spike features determining ‘host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond

“…Echoing the important role of TMPRSS2 in SARS-CoV infection, a recent study further indicated that serine proteases (e.g., TMPRSS2) but not cysteine proteases (e.g., cathepsin L) are required for SARS-CoV spread in vivo [48]. Furthermore, TMPRSS2 as well as other host enzymes, such as HAT and ADAM17, are also indicated in the shedding of human ACE2 receptor, which, in turn, was shown to promote the uptake of virus particles [49,50]. Remarkably, SARS-CoV S also contains an S2 0 cleavage site downstream of the S1/S2 boundary [51][52][53].…”