Enzyme Trypsin Facilitates SARS-CoV-2 Entry At The Cell Surface


Researchers from Gyeongsang National University-South Korea and the Gyungbuk Veterinary Service Laboratory-South Korea, have in a new study discovered that the enzyme trypsin facilitates SARS-CoV-2 entry at the cell surface.

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.

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 genome of SARS-CoV-2 encodes 16 nonstructural proteins (nsp1–nsp16); four canonical coronaviral structural proteins, named spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins; and several accessory proteins [6].

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].

In an infection experiment with SARS-CoV-2, a Vero E6 cell line overexpressing TMPRSS2 produced a higher viral titer than the parental cells, indicating the importance of this protease for the fusion capacity and infectivity of SARS-CoV-2 [11].

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].

Therefore, we investigated whether exogenous trypsin treatment enhances SARS-CoV-2 infection in Vero E6 cells and how it promotes viral replication in cultured cells.


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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