Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

Abstract

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

Fig. 1

Ben-HCl inhibits the infection of SARS-CoV and pseudotyped SARS-CoV/HIV. (A) Inhibitory effect of Ben-HCl on Vero E6 cells infected with wild-type SARS-CoV. The mean values from three independent assays are shown with standard error (SE). (B) Inhibitory effect of Ben-HCl on viral entry. 293T/ACE2 cells were infected with pseudovirus in the presence or absence of Ben-HCl, and RLU was measured. The data are shown as mean + SE of three independent experiments. (C) Correlation of entry inhibitory activity of Ben-HCl on pseudovirus infection. The data are presented as mean values of three independent experiments.

Fig. 2

In vitro cleavage of SARS-CoV S protein by Factor Xa. (A) The full-length recombinant S protein (1.55 μg) was incubated with 0.5 U Factor Xa (S + FXa) for 6 h. Untreated S protein (S only), and S protein incubated with Factor Xa and 20 mM Ben-HCl (S + FXa + Ben) for 6 h, were detected by Western blot. The cleaved S1 (∼105 kDa) and S2 (∼75 kDa) fragments, which were respectively recognized by anti-S1 (121B8), anti-S2 (102D7), and both (anti-S1 + -S2) are indicated. (B) The pseudovirus (3 ng p24) was incubated with Factor Xa (FXa) at the indicated concentrations with or without 80 mM Ben-HCl (Ben) for 2 h, and detected by Western blot using anti-S1 (121B8) and anti-S2 (119F6), respectively. (C) The pseudovirus (Ps) was incubated with 0.5 U Factor Xa (Ps + FXa), with buffer only (Ps only), or with 0.5 U Factor Xa and 80 mM Ben-HCl (Ps + FXa + Ben) for 6 h. Proteins were detected by Western blot using the same antibodies as in (B). The molecular marker (kDa) is indicated on the left.

Fig. 3

Association of pseudovirus infection with the cleavage of S protein. (A) Culture supernatant of 293T/ACE2 cells infected with pseudovirus (3 ng p24) in the presence (Ps + Ben Incub) and absence (Ps Incub) of Ben-HCl (20 mM) was collected at the indicated time points and detected for the cleavage of S protein by Western blot using anti-S1 (121B8) and anti-S2 (119F6), respectively. The pseudovirus alone (Ps alone) incubated at the same condition was included as a control. (B) After removing the pseudovirus at the indicated time points, the cells were continually cultured for 48 h and harvested for luciferase assay to determine pseudovirus infectivity, which is shown as mean + SE of RLU from three independent experiments.

Fig. 4

Expression of protease Factor Xa in 293T/ACE2 cells. (A) RT-PCR products of indicated sizes were amplified from total RNA samples extracted from 293T/ACE2 cells. The products correspond to nucleotide sequences 5–260 (255 bp), 552–889 (337 bp), and 872–1502 (630 bp) of Factor Xa transcript, respectively. The molecular marker (bp) is shown on the left. RT-PCR product of β-actin mRNA (172 bp) was amplified in the same RNA samples as an internal control. (B) Factor Xa protein expression was detected in 293T/ACE2 cell lysate by Western blot. Factor Xa of 53 kDa and β-actin of 42 kDa were recognized by their specific antibodies, respectively. The molecular marker (kDa) is indicated on the left.