The coronaviruses typically infect cells by cytoplasmic or endosomal membrane fusion, driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and the S2′ site by cellular proteases.
It has been found that exogenous trypsin serves as a medium additive that facilitates isolation and propagation of several coronaviruses in vitro.
Interestingly, trypsin enabled viral entry at the cell surface led to a more efficient infection than non-trypsin endosomal entry, suggesting that trypsin production or presence in certain organs may trigger a high level of replication of SARS-CoV-2 and cause severe tissue injury.
The study findings were published peer reviewed journal: Archives of Virology.
https://link.springer.com/article/10.1007/s00705-021-05343-0
SARS-CoV-2 is the seventh human coronavirus that has been identified, and it belongs to the subgenus Sarbecovirus of the genus Betacoronavirus in the family Coronaviridae [5]. SARS-CoV-2 is a large, enveloped RNA virus that possesses a single-stranded positive-sense RNA genome of approximately 30 kb.
The external S protein anchored on the viral envelope binds to the angiotensin-converting enzyme 2 (ACE2) receptor to mediate viral entry into host cells during SARS-CoV-2 infection [7, 8].
Like those of other coronaviruses, the S protein of SARS-CoV-2 can be functionally divided into two subunits (S1 and S2) that harbor the receptor-binding domain and the fusion domain, respectively [9]. In general, coronaviruses enter target cells via either cytoplasmic or endosomal membrane fusion, which is a primary determinant of viral infectivity.
Efficient proteolytic priming of S at the S1/S2 and S2′ sites is essential for exposing the fusion peptide and mediating membrane fusion to complete the viral entry process [10]. To accomplish S protein priming, SARS-CoV-2 uses either endosomal cysteine proteases or transmembrane protease serine 2 (TMPRSS2) [10].
Trypsin is another protease that has been shown to facilitate the fusion and infection of human and animal coronaviruses [12,13,14,15] and is essential for the propagation of swine enteric coronaviruses such as porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in vitro [16,17,18,19,20].
Discussion
Receptor binding and subsequent proteolytic priming of the S protein are prerequisites for coronavirus entry into host cells. The priming event can be mediated by various host cell proteases, including trypsin, TMPRSS2, furin, and cathepsin [29]. Trypsin has been shown previously to promote the replication of porcine coronaviruses, including the emerging/re-emerging swine coronaviruses PEDV and PDCoV, and is therefore commonly used in their isolation and cultivation [15, 17, 19].
Furthermore, protease-mediated enhancement of infection is known for SARS-CoV-1, as well as viruses belonging to other families, such as influenza and parainfluenza viruses [13]. In this study, we demonstrated that trypsin treatment enhances SARS-CoV-2 infection in cultured cells when it is added at early time points in infection (at least until 2 hpi).
The entry process of SARS-CoV-2 begins with the attachment of viral particles to the cell surface through the interaction between the viral S protein and its cellular receptor ACE2 [30]. The S protein consists of two functional subunits, S1 and S2; the former is responsible for ACE2 binding, and the latter is involved in the fusion of viral and cellular membranes.
Following endocytic uptake of SARS-CoV-2, pH-dependent proteolytic cleavage of the S protein at the S1/S2 junction is executed by the cellular serine protease TMPRSS2, thereby activating the entire fusion process [10]. Several other proteases, including trypsin, are known to facilitate the entry of SARS-CoV-1 at the cell membrane, indicating that SARS-CoV-1 can enter cells via either an endosomal or non-endosomal pathway, depending on the presence of proteases [13].
Similarly, the present study showed trypsin-mediated augmentation of SARS-CoV-2 entry at the cell surface. Thus, like SARS-CoV-1, SARS-CoV-2 can also exploit two different entry pathways, depending on the presence of trypsin. Moreover, the non-endosomal or direct entry of SARS-CoV-2 at the cell surface, mediated by trypsin, resulted in a >1-log more efficient infection than the endosomal entry pathway. Trypsin treatment before or during viral inoculation for 1 h failed to enhance infection.
Thus, we conclude that the virus requires at least 1 h for trafficking from the cell surface to bind to ACE2, suggesting the involvement of trypsin in a post-attachment step (i.e., internalization). Given that protease-aided fusion occurred after the virus was bound to the cell surface, it appears that the binding of the S protein to ACE2 might cause a conformational change that allows fusion to be triggered by a subsequent protease cleavage.
SARS-CoV-2 can replicate in the respiratory (upper and lower) and gastrointestinal tracts [9]. The results of the present study imply that trypsin-like proteases secreted in the airways and the lungs might play a role in enhancing the replication of SARS-CoV-2 in those target organs, resulting in severe tissue damage.
In particular, SARS-CoV-2 infection of pneumocytes could induce an inflammatory response that produces numerous proteases, some of which could enhance infection, resulting in a higher level of growth of SARS-CoV-2 in the lungs and therefore more tissue damage.
Severe COVID-19 pneumonia accompanied by lung injury in COVID-19 patients is associated with high plasma concentrations of multiple cytokines, a so-called cytokine storm [31], and a higher rate of virus replication could contribute to the cytokine storm by destroying a larger number of infected cells. However, we found that elastase, a major protease generated during pneumonia, had no enhancing effect on SARS-CoV-2 infection, in contrast to a previous study showing elastase-mediated enhancement of SARS-CoV-1 infection [13].
This discrepancy might be attributable to the fact that a greater than tenfold higher concentration of elastase was used in that study compared to our study. In addition, it is possible that coinfection with non- or low-pathogenic bacteria in respiratory organs favors the production of proteases, which, in turn, results in severe lung illness in SARS-CoV-2-infected patients.
Since underlying diseases play a critical role in severe COVID-19 illness in adults, certain medical conditions that dysregulate levels of trypsin or other proteases, such as chronic muco-obstructive lung diseases, might also be risk factors for poor outcomes in patients with SARS-CoV-2 infection [32, 33].
On the other hand, the small intestine, another major target organ, expresses ACE2 and TMPRSS2 and secretes a variety of proteases [10, 34]. The presence of trypsin might also result in increased multiplication of SARS-CoV-2 in the small intestine, which could be associated with the high incidence rate of diarrhea in patients with COVID-19 [35].
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