TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2

Continued high-level spread of SARS-CoV-2 has enabled an accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and in concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) dependent entry by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on cleavage by TMPRSS2. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 cleavage by TMPRSS2 led to either augmentation or attenuation of viral infectivity of pre-Omicron and Omicron lineages, respectively. Mutagenesis of the TMPRSS2 cleavage site in ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 activation of viral fusion. Our data supports the evolution of Omicron lineages towards the use of pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins. ### Competing Interest Statement The authors have declared no competing interest.