Keywords: SARS-CoV-2, renin-angiotensin system, Spike protein S1 subunit, COVID-19 pathogenesis.
The pandemic of COVID-19 disease that caused already about two millions of fatalities worldwide is caused by the coronavirus SARS-CoV-2 that is most probably transited from an animal host to humans in late 2019 [1]. The virus is closely related to the bat Coronavirus RaTG13, however, the existence of a transient host (probably a pangolin) was suggested [2]. The adaptation of SARS-CoV-2 virus to human host is believed to be mostly due to acquisition of the novel sequence of the tail spike (S) protein receptor binding domain (RBD) that efficiently recognizes human angiotensin converting enzyme 2 (ACE2). Although a related SARS-CoV coronavirus also recognizes ACE2 as its cellular receptor [1], only 8 out of 14 critical a.a. residues involved into the interaction of the ACE2 and RBD are conserved between SARS-CoV and SARS-CoV-2 viruses [2].
The coronavirus entry into the host cell requires cleavage of the S protein by the host proteases at the junction between S1 and S2 subunits [1]. This processing can take place after the virion attachment by the cell surface protease TMPRSS2 or in the lysosomal compartment after the internalization of the virus [1, 3]. The proteolytic processing of S protein allows S1 subunit to dissociate to trigger the S2 subunit rearrangement to extended post-fusion conformation required for initiation of the fusion of the viral and lysosomal membranes [4].
In some coronaviruses, however, the S protein processing may take place during the virion assembly in the Golgi compartment. Such processing requires furin protease recognition site to be present at the S1 and S2 subunits junction [3]. In SARS-CoV-2 the  insertion of 4 a.a. sequence PRRA after the a.a. 675 in S protein that lead to the formation of the furin cleavage site RRAR [2]. This leads to efficient processing of the S protein in the virus-producing cell [3, 5]. The pre-processing of the spike protein before the virus release makes the infection more efficient [3] and potentially may allow part of the virus particles to penetrate into the host cells directly at the plasma membrane without entering to the lysosomal compartment [6]. More efficient host cell entry taken together with higher affinity of its RBD (compared to RBD of SARS-CoV that lacks efficient furin processing site) may compensate SARS-CoV-2 for decreased availability of the RBDs for ACE2-receptor binding [3]. Indeed, on most of the SARS-CoV-2 S protein trimers two out of three RBDs remain in closed conformation in which they are shielded from both host immunity factors (e. g. antibodies) and from receptor recognition, while in SARS-CoV virus all three RBDs are in “open” conformation on most of the spikes[7].
As in any virus pneumonia, the pathology of COVID-19, as well of SARS, is believed to be mainly due to the virus killing of the lung epithelial cells, and, even to larger extent, due to immune-mediated mechanisms [1, 8].
However, the recognition of ACE2 as the receptor by both SARS-CoV and SARS-CoV-2 raised a hypothesis that in addition to the aforementioned mechanisms (that may be further enhanced by a secondary bacterial infection) the pathology of COVID-19 and of SARS may in large extent rely on the virus-induced disbalance of the renin-angiotensin system (RAS)[9, 10].
 ACE2 is a cell-surface metalloprotease (carboxypeptidase) that converts angiotensin I decapeptide into angiotensin(1-9) nonapeptide, in contrast to ACE enzyme, that produced out of angiotensin I the physiologically active angiotensin II nonapeptide. Angiotensin(1-9) can be converted by ACE enzyme into angiotensin(1-7) heptapeptide. Angiotensin II also may be processed into angiotensin(1-7) by ACE2[9, 10]. The physiological activity of angiotensin II leads to vasoconstriction and increased arterial pressure but it also promotes inflammation reactions, blood coagulation and thrombosis, fibrosis capillary permeability and edema. Angiotensin1-7 has basically opposite effect decreasing inflammation, thrombosis, fibrosis and causes vasodilatation [9, 10]. Therefore, upregulation of ACE and/or downregulation of ACE2 lead to increased pulmonary damage.
It has been demonstrated that SARS-CoV infection decreases ACE2 expression in murine model [9-11] suggested that virus attachment to ACE2 molecules lead to their removal from the cell surface via co-endocytosis with the virus thus diminishing the ACE2 activity. This leads to the imbalance between angiotensin II and angiotensin(1-7) in the lung tissue and to increases thrombosis and pulmonary damage [9, 10].
From our point of view, the direct mechanical removal of the ACE2 molecules by the virion attachment is unlikely to contribute significantly to the overall activity of the enzyme in the lungs. Such an effect would require simultaneous virus attachment to large fraction of the ACE2 producing cells (such as alveocytes II). We believe that at that high virus load the direct damage of the epithelial cells would not be compatible with the patient survival, though the reported mortality even in severe COVID-19 cases is moderate [1, 8].
Interestingly, in [11] study the reduction of ACE2 in mice could be induced not only by SARS-CoV infection but also by the recombinant SARS-CoV spike protein. The mice pre-treated (i.p.) with this spike protein did not show significant pathology but if these animals were acid instilled, the spike protein pre-treatment lead to increased severity of the lung damage [11].
Therefore, the spike protein if present in significant molar excess in respect to the viral particles may in fact mediate the down regulation of ACE2 and RAS imbalance. It has been shown that S proteins of murine coronaviruses and of SARS-CoV are indeed delivered onto the cell surface, presumably, as a side product of the virus assembly and release process. These molecules may induce some physiological effect such as micropinocytosis and/or membrane fusion of the neighbor cells [6], however S protein been a membrane protein, remains attached to the infected cell membrane. It worth to mention that S protein is also present on the viral particles released from the infected cells into the medium.
It has been recently demonstrated by direct cryo-electron and negative-stain electron microscopy [12] that on the in vitro cultured SARS-CoV-2 particles retaining their infectivity most of S protein trimers are in the post-fusion conformation. In the other words, the S1 subunits dissociation took place before the actual receptor binding (apparently the biological activity of these virions was assured by the remaining fraction of S trimers that did not yet shot in vain). Although in the conditions of the study, the S1 subunits dissociation could be triggered by the conditions during the gradient centrifugation used to purify the virus [12]. Nevertheless, this result demonstrates that in SARS-CoV-2 processed spikes S1 subunit dissociation can be triggered by relatively mild conditions that are compatible with the virus viability, or occur spontaneously. We hypothesize that substantial amount of free soluble S1 subunits may be shed from the infected cells and virions (Fig 1).