Contribution of trypsin to SARS-CoV-2 infection
To explore the impact of trypsin on viral replication, Vero E6 cells were incubated with trypsin for 1 h at the following concentrations (0, 1.25, 2.5, 5, and 10 µg/ml) and then mock-infected or SARS-CoV-2 (P3)-infected at an MOI of 1 for 1 h. The infected cells were washed once with PBS and cultivated in the cell growth medium deprived of trypsin. According to the MTT cell viability assay, none of the trypsin dosages examined in this study resulted in measurable degrees of cell death (data not shown). SARS-CoV-2 replication was initially measured by monitoring the intensity of cytopathic effect (CPE) and was confirmed by IFA using the anti-N protein MAb at 24 hpi. As shown in Fig. 1A, treating cells with trypsin before virus infection had no effect on SARS-CoV-2 infectivity at any concentration tested compared to the untreated control. We further quantified viral production using virus titration at 24 hpi. The virus yields remained unchanged, with mean titer ranges of 106.53–106.73 TCID50/ml regardless of the presence or absence of trypsin before infection (Fig. 1B). Furthermore, SARS-CoV-2 propagation in cell culture was unaffected by adding exogenous trypsin during the 1 h-inoculation period (Fig. 1C and D). These results revealed that trypsin treatment before or during infection did not influence SARS-CoV-2 replication.
Next, we investigated whether SARS-CoV-2 infectivity is enhanced by supplementing trypsin after SARS-CoV-2 infection. Vero E6 cells were subjected to SARS-CoV-2 infection at an MOI of 1 for 1 h without trypsin and then propagated in the FBS-free medium with trypsin at the following concentrations (0, 1.25, 2.5, 5, and 10 µg/ml). Unless otherwise specified, trypsin was present throughout the infection period. CPE observations and IFA analyses revealed that when the virus-infected cells were cultivated in the presence of trypsin, the virus infectivity increased (Fig. 1E). When exogenous trypsin was added at post-inoculation, virus titers were considerably enhanced compared to viral propagation without trypsin (Fig. 1F). The highest viral titer (107.52 TCID50/ml) was achieved in the presence of 5 µg/ml trypsin, which was 1-log higher than the control mean titer (106.67 TCID50/ml). Additionally, Vero E6 cells were inoculated with ten-fold serially diluted SARS-CoV-2 and maintained in the presence or absence of trypsin (5 µg/ml), and IFA was used to visualize the infected cells (Fig. 2). The results revealed that Vero E6 cells propagated with trypsin displayed a >10-fold higher number of SARS-CoV-2-infected cells than those cultured without trypsin addition. The virus growth medium (high-glucose DMEM supplemented with penicillin-streptomycin and 5 µg/ml trypsin) was then used in all subsequent experiments unless otherwise indicated. We also tested the effect of another protease, elastase, on SARS-CoV-2 under the same experimental conditions. However, no apparent effect of elastase on SARS-CoV-2 replication was detected in cell culture when cells were treated with elastase at non-cytotoxic concentrations (5–10 µg/ml) before, during, or after infection (Fig. 3). These data indicated that the presence of trypsin at post-virus inoculation promotes the replication of SARS-CoV-2 in a protease type-dependent manner.
To establish the point at which trypsin is present during SARS-CoV-2 infection, we added trypsin to Vero E6 cells at different intervals post-infection. At 24 hpi, the extent of viral replication was assessed directly by virus titration. As presented in Fig. 4, the addition of trypsin at up to 2 hpi (i.e., 3 h after inoculation) resulted in a considerable enhancement of SARS-CoV-2 infectivity. However, when trypsin was introduced at or after 4 hpi, little or no increase in SARS-CoV-2 propagation was observed. These findings established that trypsin must be present during the early stages of viral infection in order to effectively enhance SARS-CoV-2 infection. Taken together, our results suggest that trypsin participates in the entry pathway of SARS-CoV-2.
Involvement of trypsin in the virus internalization
We next explored whether trypsin facilitates the entry process of SARS-CoV-2 in cultured cells. To accurately corroborate the parameters of the viral internalization assay necessary for monitoring SARS-CoV-2 entry, we used a proteinase K infectious assay that assesses the rate of virus attachment and penetration by quantifying productive bound or internalized virus particles, respectively. To determine the effectiveness of proteinase K treatment in removing associated viruses, Vero E6 cells were infected with SARS-CoV-2 for 1 h at 4°C and then treated with proteinase K at the specified doses for 45 min at 4°C. The quantity of virus bound was measured using qRT-PCR to determine the SARS-CoV-2 genome copy number. As demonstrated in Fig. 5A, proteinase K treatment had a substantial effect on the quantity of bound virions at the cell surface, suggesting that virus linked to the cells is efficiently removed by proteinase K treatment.
We then studied the two phases of viral entrance (attachment and penetration) in the presence of trypsin by virus binding and internalization assays. Vero E6 cells were inoculated with SARS-CoV-2 at 4°C for 1 h to enable only virus attachment and then maintained at 4 or 37°C in the presence of trypsin to limit or permit virus internalization, respectively. Following that, proteinase K was used to remove any remaining virus particles from the cell surface. Serial dilutions of infected cells were performed on fresh Vero E6 cell monolayers, and the viral titers were determined (Fig. 5B). Regardless of the presence or absence of trypsin, the viral titers were equivalent among cells treated at 4°C to allow virus binding but inhibit penetration, However, virus production was considerably enhanced in cells maintained at 37°C with trypsin to allow virus internalization to progress, compared to the vehicle control without trypsin under the same condition. These findings suggest that trypsin accelerates SARS-CoV-2 internalization.
SARS-CoV-2 entry at the cell surface facilitated by trypsin
To test whether SARS-CoV-2 enters cells by a pH-dependent endosomal pathway, we first evaluated the effect of BafA1, a lysosomotropic agent, on SARS-CoV-2 replication by virological analyses. Vero E6 cells were treated with BafA1 at concentrations of 0.1 and 0.5 µM or DMSO (0.5%) as a vehicle control before, during, or after infection. The MTT assay revealed that cellular cytotoxicity did not occur in drug-treated cells at the concentrations used in the study (data not shown). Viral production was measured by IFA using anti-N protein MAb and confirmed by virus titration at 24 hpi (Fig. 6). Treating cells with BafA1 before or during infection significantly attenuated SARS-CoV-2 N expression (Fig. 6A and C) and further suppressed viral infection (Fig. 6B and D) in a dose-dependent manner. By contrast, virus propagation remained unchanged, regardless of the presence or absence of BafA1 after infection (Fig. 6E and F). Nevertheless, these results suggest that SARS-CoV-2 entry occurs through an endosomal pathway.
We further investigated the effectiveness of trypsin in facilitating SARS-CoV-2 entry directly at the cell surface. Vero E6 cells treated with BafA1 at a concentration of 0.5 µM were inoculated with SARS-CoV-2 at an MOI of 1 and adsorbed at 4°C for 30 min to block the virus entry into cells. The cells were then treated with different proteases at room temperature for 5 min and maintained at 37°C for 6 h, and virus internalization was calculated by qRT-PCR (Fig. 7). Trypsin greatly facilitated SARS-CoV-2 entry, whereas elastase did not influence viral entry (Fig. 7A). Trypsin treatment of cells prior to viral infection had no influence on SARS-CoV-2 internalization (Fig. 7B), showing that the effects of trypsin on cells are irrelevant for this infection. These results imply that SARS-CoV-2 entry occurs via a non-endosomal, direct fusion in the presence of trypsin, which cleaves the fusion-inducing S protein.
Treatment with high concentrations (10–75 µg/ml) of trypsin mediated the enhancement of virus entry or replication compared with the normal infection without trypsin (Fig. 7A; compare bars 6–8 with bar 1 from left). The replication kinetics of SARS-CoV-2 were then compared in cells treated with BafA1 (0.5 µM) and a high concentration (75 µg/ml) of trypsin to those in virus-infected cells maintained in the absence of BafA1 and trypsin. At any point during the early stage of infection, trypsin-treated cells produced substantially more virus (Fig. 7C).
Phenotypic and genotypic characterizations of SARS-CoV-2 serially passaged in the presence or absence of trypsin
We serially propagated viruses in vitro up to 50 passages in the presence or absence of trypsin and generated virus stocks every ten passages labeled P10, P20, P30, P40, and P50. Like the parental virus (P3), all SARS-CoV-2 strains cultured in the absence of trypsin produced CPE typical of viral infection, such as cell rounding, clumping together in clusters, and detachment, in infected Vero E6 cells (Fig. 8, left panels). However, trypsin-adapted strains could induce different patterns of CPE that included cell fusion and multi-nucleated cells or syncytia formation in infected cells (Fig. 8, right panels). The vacuolation and syncytia were larger and more predominant in cells infected with SARS-CoV-2 strains that were more consecutively passaged in the presence of trypsin. As a consequence, the size of syncytia increased progressively with the serial passage number, and the high-passage P50 virus generated the prevalent syncytia with many more nuclei than the low-passage P10 virus in the presence of trypsin (Fig. 8; compare panel n with panel j).
To examine the phenotypic characteristics of serially passaged SARS-CoV-2 strains in vitro, we evaluated the one-step growth rates of representative P3 and P10 strains cultured with or without trypsin (Fig. 9). The parental P3 virus without trypsin addition had the highest level, showing a peak titer of 105.57 TCID50/ml at 24 hpi, after which its growth declined (Fig. 9A). By contrast, the growth kinetics of the P3 virus were significantly promoted by trypsin. At 12 hpi, the P3 virus in the presence of trypsin caused a rapid increase in virus titers, which were maintained until 48 hpi, ranging from 106.33 to 107 TCID50/ml. Furthermore, the cell-adapted viruses serially passaged under the trypsin-free condition showed similar growth curves and virus titers to those of the P3 virus, with an apex at 24 hpi (Fig. 9B). Despite the comparable growth curves of the P3 and P10 strains, the trypsin-adapted virus grew faster and produced higher titers as the passage number advanced. In particular, cells infected with the trypsin-adapted P10 virus reached virus titers of 107.93 TCID50/ml at 12 hpi and continued to produce up to 48 hpi, which was maximally 100-fold higher than that of the parental or passaged strain without trypsin addition. Compared to the P10 virus cultured in the presence of trypsin, the growth patterns were comparable among further trypsin-adapted P20–P50 strains passaged under trypsin addition (data not shown). These results demonstrated that trypsin is able to promote SARS-CoV-2 infection by facilitating cell-to-cell fusion.
To evaluate the genomic alterations that may have occurred during in vitro serial passages in Vero E6 cells in the presence or absence of trypsin, we determined the full-length nucleotide sequences of the parental P2 and its derived passages, P3–P50, using Sanger sequencing and RACE. The sequence data divulged no mutations in the P3 virus propagated in the presence (+) or absence (−) of trypsin compared with the original KCDC03 P2 strain. Although the 5′-and 3′-untranslated regions (UTRs) remained unchanged during in vitro serial passages in Vero E6 cells, virus mutations emerged in the protein-coding regions and increased gradually over time. At the nucleotide level, the genomes of P3(+) or P3(−) shared a high degree of similarity (99.94–99.99%) with the respective cell-adapted strain. In comparison to the parental P3(+) or P3(−) strain, the numbers of nucleotide/amino acid substitutions increased in direct proportion to the number of the in vitro passages, resulting in the highest and lowest identities with each respective P10 and P50 (Table 1).
Table 1
The number of nucleotide and amino acid changes between the parental KNU-SARS-CoV-2 P3 and cell-adapted viruses
UTRs/ORFs | Encoded proteins | No. of nucleotide/amino acid changes (No. of amino acid deletion) |
P10(−a) | P20(−) | P30(−) | P40(−) | P50(−) | P10(+b) | P20(+) | P30(+) | P40(+) | P50(+) |
5′ UTR | | -c | - | - | - | - | - | - | - | - | - |
ORF1a | nsp1 | - | - | 3/1d | 3/1 | 3/1 | - | - | - | - | - |
| nsp2 | - | - | - | - | - | - | - | - | - | - |
| nsp3 | - | - | - | - | - | - | - | - | - | - |
| nsp4 | - | - | - | - | - | - | - | 1/1 | 1/1 | - |
| nsp5 | - | - | - | - | - | - | - | 1/1 | - | - |
| nsp6 | - | - | - | - | - | - | - | - | 1/1 | 1/1 |
| nsp7 | - | - | - | - | - | - | - | - | - | 1/0 |
| nsp8 | - | - | - | - | - | - | - | - | - | - |
| nsp9 | - | - | - | - | - | - | - | - | - | - |
| nsp10 | - | - | - | - | - | - | - | - | - | - |
| nsp11 | - | - | - | - | - | - | - | - | - | - |
ORF1b | nsp12 | - | - | - | - | - | - | - | - | - | - |
| nsp13 | - | - | - | - | - | - | - | - | - | 1/1 |
| nsp14 | - | - | - | - | - | - | - | - | - | - |
| nsp15 | - | - | - | - | - | - | - | - | - | - |
| nsp16 | - | - | - | - | - | - | - | - | - | - |
ORF2 | S | 2/2 | 3/3 | 5/5 | 5/5 | 5/5 | 2/2 | 6/5 | 4/3 | 4/3 | 6/5 |
ORF3a | ORF3a | - | - | - | - | - | - | - | - | - | - |
ORF4 | E | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 | - | - | - | - | - |
ORF5 | M | 1/1 | 1/1 | - | - | - | - | - | - | 1/1 | 1/1 |
ORF6 | ORF6 | - | - | - | - | - | 1/1 | 1/1 | 1/1 | 1/1 | 1/1 |
ORF7a | ORF7a | - | - | - | - | - | - | - | - | - | - |
ORF7b | ORF7b | - | - | - | - | - | - | - | - | 4/13e | 4/13 |
ORF8 | ORF8 | - | - | - | - | - | - | - | 1/1 | 1/1 | 1/1 |
ORF9 | N | 1/2f | 1/2 | 1/2 | 1/2 | 1/2 | - | - | - | - | - |
ORF10 | ORF10 | - | - | - | - | - | - | - | - | - | - |
3′ UTR | | - | - | - | - | - | - | - | - | - | - |
Total number | | 5/6 (2) | 6/7 (2) | 10/9 (3) | 10/9 (3) | 10/9 (3) | 3/3 | 7/6 | 8/7 | 13/21 (13) | 16/23 (13) |
aSARS-CoV-2 strains passaged in the absence of trypsin |
bSARS-CoV-2 strains passaged in the presence of trypsin |
cNo mutations |
dThe deletion of three nucleotides (ATG) covering the codon for one amino acid (methionine) |
eThe deletion of 13 amino acid residues by an early termination due to a C92A substitution in ORF7b |
fThe deletion of two amino acid resided by an early termination due to a C1252T substitution in N |
Interestingly, the number and location of the amino acid (aa) changes differed between the cell culture-passage strains in the presence or absence of trypsin (Fig. 10). The 50th-passage strain without trypsin addition contained 9 aa mutations, including 3 aa deletions (DELs), whereas the P50(+) virus had 23 aa variations, including 13 aa DELs. The 9 aa mutations in the P50(−) virus were distributed in open reading frames (ORFs) 1a, 2, 4, and 9 encoding nsp1, S, E, and N, respectively. Among those, 5 aa changes were accumulated in the S protein of P50(−). Notably, 1 aa mutation (R685S) was found in an S1/S2 furin cleavage site (FCS) consisting of multiple basic amino acids (681PRRAR685) (Table 2), which was found since P10(−). One (M) and two (QA) DELs emerged independently in nsp1 and N during the cell culture passages in the absence of trypsin, the former in P30(−) and the latter in P10(−) (Table 2). The M-DEL at position 85 in nsp1 resulted from a three-nucleotide (AUG) DEL covering the codon for methionine (M) at positions 253–255 in ORF1a (490–492 at the genome level). At positions 418 and 419 in N, the QA-DEL arose from a C to T substitution (C1252T) at position 1252 in ORF9 (29,497 at the genome level). This change resulted in the modification of CAG coding for glutamine (Q) to TAG terminator sequence at positions 1252–1254 in ORF9 (29,497–29,499 at the genome level), causing a premature termination directly removing two QA residues from the C-terminal end of N.
Table 2
Summary of amino acid mutations during in vitro serial passages
ORFs | Encoded proteins (aa length) | aa positiona | | Mutation at the indicated passage number |
P3 | P10(−b) | P20(−) | P30(−) | P40(−) | P50(−) | P10(+c) | P20(+) | P30(+) | P40(+) | P50(+) |
ORF1a | nsp1 (180) | 85 | M | | | DEL | DEL | DEL | | | | | |
| nsp4 (500) | 106 | L | | | | | | | | F | F | |
| nsp5 (306) | 302 | G | | | | | | | | R | | |
| nsp6 (290) | 192 | M | | | | | | | | | R | R |
ORF1b | nsp13 (601) | 413 | T | | | | | | | | | | I |
ORF2 | S (1273) | 68 | I | | | | | | | R | R | R | R |
| | 178 | D | N | N | N | N | N | | | | | |
| | 185 | K | | | | | | N | N | | | |
| | 186 | F | | | | | | V | V | | | |
| | 484 | E | | D | D | D | D | | | | | |
| | 655 | H | | | Y | Y | Y | | | | | |
| | 685 | R | S | S | S | S | S | | | | | |
| | 813 | S | | | I | I | I | | | | | |
| | 814 | K | | | | | | | | | | R |
| | 949 | Q | | | | | | | | | | R |
| | 960 | N | | | | | | | I | I | I | I |
| | 991 | V | | | | | | | A | A | A | A |
ORF4 | E (75) | 22 | A | V | V | V | V | V | | | | | |
ORF5 | M (222) | 125 | H | Y | Y | | | | | | | Y | Y |
ORF6 | ORF6 (61) | 2 | F | | | | | | L | L | L | L | L |
ORF7b | ORF7b (43) | 31–43 | S–Ad | | | | | | | | | DEL | DEL |
ORF8 | ORF8 (121) | 67 | S | | | | | | | | F | F | F |
ORF9 | N (419) | 418/419 | QA | DEL | DEL | DEL | DEL | DEL | | | | | |
aAmino acid position numbering on the basis of the genomic sequence of SARS-CoV-2 P3 strain |
bSARS-CoV-2 strains passaged in the absence of trypsin |
cSARS-CoV-2 strains passaged in the presence of trypsin |
dDeleted amino acid residues SLELQDHNETCHA in ORF7b |
The 23 aa variations in the 50th passage in the presence of trypsin were dispersed randomly in ORFs 1a, 1b, 2, 5, 6, 7b, and 8 encoding nsp6, nsp13, S, and M, as well as accessory proteins, respectively (Table 1). Although the number (five) of aa mutations in the S protein of P50(+) was identical to that in P50(−), their positions in the P50(+) S protein were completely distinct from those in P50(−) (Fig. 10). Intriguingly, 13 aa DELs occurred at positions 31–43 in ORF7b of P40(+) and were maintained until the 50th cell culture passage in the presence of trypsin (Table 2). The S–A-DEL resulted from a C to A substitution (C92A) at position 92 in ORF7b (27,819 at the genome level). This mutation altered the sequence TCA, encoding serine (S), to a TAA termination codon at positions 91–93 in ORF7b (27,818–27,820 at the genome level), leading to an early termination eliminating 13 aa residues from the C-terminus of ORF7b.