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 (Chen, Liu &
Guo, 2020), 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 (Zhang, Wu & Zhang, 2020).
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
(Chen, Liu & Guo, 2020). 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 (Chen, Liu
& Guo, 2020; Shang et al., 2020). 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 (Walls et
al., 2017).
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 (Shang et al., 2020). 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 (Zhang, Wu & Zhang, 2020).
This leads to efficient processing of the S protein in the
virus-producing cell (Hoffmann, Kleine-Weber & Pöhlmann, 2020; Shang et
al., 2020). The pre-processing of the spike protein before the virus
release makes the infection more efficient (Shang et al., 2020) 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 (Freeman, Peek, Becker, Smith & Denison, 2014).
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 (Shang et al., 2020). 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 (Wrapp et al., 2020).
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
(Chen, Liu & Guo, 2020; Matricardi, Dal Negro & Nisini, 2020).
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)(Gheblawi et al., 2020; Verdecchia, Cavallini, Spanevello &
Angeli, 2020).
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) heptapetide. Angiotensin II also may be
processed into angiotensin(1-7) by ACE2(Gheblawi et al., 2020;
Verdecchia, Cavallini, Spanevello & Angeli, 2020). 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 (Gheblawi
et al., 2020; Verdecchia, Cavallini, Spanevello & Angeli, 2020).
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 (Gheblawi et al., 2020; Kuba et al., 2005;
Verdecchia, Cavallini, Spanevello & Angeli, 2020) 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
(Gheblawi et al., 2020; Verdecchia, Cavallini, Spanevello & Angeli,
2020).
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 (Chen, Liu & Guo, 2020;
Matricardi, Dal Negro & Nisini, 2020).
Interestingly, in (Kuba et al., 2005) 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 (Kuba et al., 2005).
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 (Freeman,
Peek, Becker, Smith & Denison, 2014), 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 (Liu et al., 2020) 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 (Liu et al., 2020).
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).