Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS. Analysis of SARS-CoV spike glycoprotein (S) using recombinant plasmid and virus infections demonstrated that the S-precursor (proS) exists as a approximately 190 kDa endoplasmic reticulum form and a approximately 210 kDa Golgi-modified form. ProS is subsequently processed into two C-terminal proteins of approximately 110 and approximately 80 kDa. The membrane-bound proprotein convertases (PCs) furin, PC7 or PC5B enhanced the production of the approximately 80 kDa protein. In agreement, proS processing, cytopathic effects, and viral titers were enhanced in recombinant Vero E6 cells overexpressing furin, PC7 or PC5B. The convertase inhibitor dec-RVKR-cmk significantly reduced proS cleavage and viral titers of SARS-CoV infected cells. In addition, inhibition of processing by dec-RVKR-cmk completely abrogated the virus-induced cellular cytopathicity. A fluorogenically quenched synthetic peptide encompassing Arg(761) of the spike glycoprotein was efficiently cleaved by furin and the cleavage was inhibited by EDTA and dec-RVKR-cmk. Taken together, our data indicate that furin or PC-mediated processing plays a critical role in SARS-CoV spread and cytopathicity, and inhibitors of the PCs represent potential therapeutic anti-SARS-CoV agents.

Fig. 1

Processing of the SARS-CoV spike glycoprotein precursor. (A) Schematic representation of the S-protein. Arrows point to potential cleavage sites (sites A and B). The signal peptide (SP), S1/S2 domains, heptad repeats (HR1, HR2), cytosolic tail (CT), and V5-epitope (V5) are illustrated. Underneath the spike scheme is an amino acid (aa) alignment of group II coronavirus cleavage sites with the SARS-CoV S. Basic aa are emphasized in bold. Downward arrows indicate group II proS identified cleavage sites. (B) FD11 cells stably expressing either the vector alone or a defined convertase were transiently transfected with the spike glycoprotein cDNA. At 48 h post-transfection with the designated constructs, cell lysates were V5-immunoprecipitated, proteins were resolved by 7% SDS–PAGE, and analyzed by Western blot with a V5 antibody. (C) Vero E6 cells stably expressing the PCs were infected with SARS-CoV, control (CTL) representing uninfected cells. After virus adsorption at a multiplicity of infection (MOI) of one for 1 h, inoculums were removed. Pulse-labeled cell lysate proteins were HMAF–immunoprecipitated and resolved by 7% NuPAGE. Proteins were visualized by autoradiography. Asterisk (∗) denotes increased spike processing.

Fig. 2

The membrane-bound PCs increase SARS-CoV titers and their inhibition by dec-RVKR-cmk reduces SARS-CoV titers. (A) Cells were infected as in Fig. 1C. Twenty-four hours post-infection, clarified cell supernatants were titrated. Serial dilutions of the virus containing supernatant were added to a fresh monolayer of Vero E6 cells followed by agar overlay. The plaques were visualized by neutral red staining. (B) Vero E6 cells were infected as in Fig. 1C, except that different doses of dec-RVKR-cmk were added to the media after inoculum removal. Viruses were titrated as described in (A). These data are representative of at least four independent experiments and are presented as average with the corresponding standard deviation.

Fig. 3

PC-CMK inhibits SARS-CoV spread and CPE. Vero E6 cells were infected with SARS-CoV (MOI = 0.5) as in Fig. 1C. Upon removal of the virus fresh media containing 60 μM dec-RVKR-cmk were added to the cells. The onset of CPE was monitored by microscopic observation. Left panel pictures were taken 24 h post-infection. At 15 h post-infection, another set of cells were permeabilized and viral antigens were probed with HMAF and stained with a fluorescein-conjugated secondary antibody (right panel).

Fig. 4

Biosynthetic analysis of PC-inhibition of SARS-CoV S-processing. (A) Vero E6 cells were infected as described in Fig. 1C. Cells were then incubated with media containing the indicated concentrations of dec-RVKR-cmk for 16 h. Labeling and electrophoresis were performed as in Fig. 1D, except that starvation and pulse labeling were performed in the presence of indicated amounts of dec-RVKR-cmk. (B) Percentage inhibition of ∼110 and ∼80 kDa CTF levels were quantified from the Phosphor Imager acquired data of (A). (C) Infections and dec-RVKR-cmk treatments were done as in (A), except that the samples were chased with cold media for 3 h. CTL lane indicates an uninfected control treated with 60 μM dec-RVKR-cmk. At the end of the 3 h chase period, media were immunoprecipitated with the antiserum HMAF.

Fig. 5

Processing of proS mutants. (A) Wild type (WT) and mutant proS were transfected in FD11 cells stably expressing furin. (B) Stable FD11-furin cells were transfected with either proS-V5 or proS-(RRKR₆₆₇)-V5 mutant. Immunoprecipitation and Western blot were performed as in Fig. 1B.

Fig. 6

In vitro digestion of proS QS1 peptide by recombinant furin. (A) The internally quenched fluorogenic peptide QS1 was incubated with recombinant furin in the absence or presence of EDTA and dec-RVKR-cmk (CMK) as indicated. Crude reactions were submitted to RP-HPLC equipped with consecutive online monitoring of ultraviolet (UV) absorbance followed by fluorescence emission. Chromatograms of QS1 were produced by the simultaneous monitoring of UV absorbance (left panel) and fluorescence emission (right panel). Control indicates undigested QS1 that showed a single major peak with a retention time (R_t) of 34.3 min. The asterisk marks a peak not specific to furin digestion. Note in the furin digest chromatogram (furin) the disappearance of the 34.3 min peak and appearance of a fluorescent N-terminal (R_t = 19.6 min) and a non-fluorescent C-terminal (R_t = 31.2 min) products. These processing peptides were not detected with dec-RVKR-cmk or EDTA. (B) Mass spectrometry identified each peptide: undigested peptide showed peaks at an m/z of 1841 (M + Na)⁺, 1819 (M + H)⁺, 1803 (M + H–NH₂)⁺, and 1787 (M + H–2NH₂)⁺. The N-terminal product exhibited an m/z of 1036 (M + H)⁺ and 1020 (M + H–NH₂)⁺. The C-terminal product displayed an m/z of 801 (M + H)⁺ and 823 (M + Na)⁺, indicating cleavage after Arg₇₆₁.