Abstract
Continued high levels spread of SARS-CoV-2 globally enabled accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in ACE2 and TMPRSS2 use 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 or non-cleavage of ACE2 by TMPRSS2 activity. In engineered cells overexpressing ACE2 and TMPRSS2, ACE2 was cleaved by TMPRSS2 and this led to either augmentation or progressive attenuation of pre-Omicron and Omicron lineages, respectfully. In contrast, TMPRSS2 resistant ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 spike activation. Therefore, our data support the tropism shift of Omicron lineages to be a function of evolution towards the use of uncleaved pools of ACE2 with the latter consistent with its role as a chaperone for many tissue specific amino acid transport proteins.
A. &B. ACE2 cleaved pool model to reconcile the evolving entry requirements of SARS CoV-2 and changes in viral tropism in vivo. A. As observed for SARS CoV-1 and early SARS CoV-2 lineages, cleavage of ACE2 by TMPRSS2 augments cellular entry by enabling TMPRSS2 Spike S2 activation to proceed. In primary lower respiratory this would proceed to finely regulate ACE2 as part of the Renin Angiogensin System (RAAS). In other settings where ACE2 is not cleaved by TMPRSS2, entry still proceeds. In both ACE2 pools, TMPRSS2 activation can proceed (“on” conformation) B. For Omicron lineages, cleavage of ACE2 by TMPRSS2 is an “off” switch for latter TMPRSS2 Spike S2 activation. Whilst this supports attenuation in tissue where ACE2 is cleavage sensitive due to its role in RAAS (e.g. lung), replication in other tissue has been observed to proceed. Independent of RAAS, ACE2 can also act as a chaperone for several tissue specific amino acid transporters which engage at and around the TMPRSS2 cleavage site. To mimic lack of ACE2 cleavage in this setting, we mutated the ACE2-TMPRSS2 cleavage site and revealed settings where ACE2 is not subject to TMPRSS2 cleavage is a “on” switch for latter TMPRSS2 Spike S2 activation. C. Through generating engineered lines that were biased for TMPRSS2 cleavage or expressed ACE2 that was TMPRSS2 resistant, we mapped the evolution of ACE2 and TMPRSS2 use across the first three years of the pandemic. In early clades, TMPRSS2 cleavage of ACE2 augmented ACE2/TMPRSS2 viral entry. This augmentation steadily decayed and led to attenuation upon the arrival of omicron BA.1 with continued attenuation observed now in XBB omicron lineages. Over the same time, TMPRSS2 cleavage resistant ACE2 has supported TMPRSS2 activation entry across all SARS CoV-2 lineages equally.
Competing Interest Statement
The authors have declared no competing interest.