Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection

Abstract

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ∼80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.

FIGURE 1.

CCHFV NP is cleaved during Bax-induced apoptosis. A, map of the entire NP. The amino acid sequence of residues 240–300 of NP is shown. The DEVD motif is indicated, and the putative cleavage site between residues 269 and 270 is indicated. B, Vero cells were transiently transfected with plasmids encoding N-terminal c-Myc-tagged full-length NP or residues 1–269 of NP. To induce apoptosis, co-transfection with Bax plasmid was performed. The cells were harvested 24 h after transfection, and the expressions of the transfected proteins were analyzed by Western blot analysis with anti-Myc and anti-FLAG antibodies. Expressions of endogenous calnexin and PARP were also analyzed by Western blot analysis. C, a similar experiment was performed with plasmids encoding C-terminal c-Myc-tagged full-length NP or residues 270 to 482 of NP. *, an unknown endogenous protein detected by the anti-Myc antibody. aa, amino acids.

FIGURE 2.

The DEVD motif, in CCHFV NP, is a subject for caspase-3 cleavage. A, BHK cells were infected with recombinant SFV-expressing CCHFV NP. Lysates from mock-infected or SFV NP-infected cells were harvested at different times postinfection and analyzed for CCHFV NP by Western blot. The arrows show full-length NP (56 kDa) and the cleavage product (30 kDa). B, cell lysates were further analyzed for PARP by Western blot (the 116 kDa band represents uncleaved PARP, and the 85 kDa band represents cleaved PARP). C, BHK cells were infected with recombinant SFV expressing native CCHFV NP or the mutated form (at positions 266 and 269) of NP. Lysates from infected cells were harvested at different period of times postinfection and analyzed for CCHFV NP by Western blot.

FIGURE 3.

A smaller, 30-kDa fragment of NP appears at a late time postinfection. A549, HUVEC, and SW13 cells were infected with CCHFV at MOI = 1. The culture medium containing detached cells was harvested at 24, 48, and 72 h postinfection and analyzed for the expression pattern of NP by Western blot. Several lanes from separate gels have been merged in this figure.

FIGURE 4.

Programmed cell death is induced during CCHFV infection. A, SW13 cells were mock-infected or infected with CCHFV (MOI = 1 or 10). Mock-infected and CCHFV-infected cells were fixed for 1 h with 4% paraformaldehyde (pH 7.4) at room temperature. Cells were then incubated with permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate. Labeling of DNA strand breaks was performed with the TMR-red cell death detection kit. Values given are the average of three independent experiments. Error bars, S.D. B, SW13 cells were mock-infected or infected with CCHFV (MOI = 1). Cells were stained for Annexin V as described under “Experimental Procedures.” Values given are the average of three independent experiments. Error bars, S.D. Significance is illustrated with p < 0.001 (**) and p < 0.0001 (***). hpi, hours postinfection.

FIGURE 5.

Caspase-3 is activated at late time post CCHFV infection. SW13 cells were infected with CCHFV (MOI = 1). At 72 h postinfection, the culture media containing detached cells (A) and the attached cells (B) were harvested separately. The lysates were analyzed for NP and caspase-3, and calnexin was used as a loading control.

FIGURE 6.

Inactivation of caspase-3 inhibits cleavage of NP. A and B, SW13 cells were infected with CCHFV (MOI = 1). After 1 h, cells were mock-treated or treated with Z-FA-fmk or Z-DEVD-fmk. The culture media containing detached cells and the attached cells were collected separately at 72 h postinfection and analyzed for NP (A) and PARP (B) by Western blot. C, mock-infected MCF-7, stable MCF-7 caspase cells, and SW13 cell lysate were analyzed for the presence of caspase-3 by Western blot. D, MCF-7 and stable MCF-7-caspase-3 cells infected with CCHFV (MOI = 1). The culture media containing detached cells and the attached cells were collected separately at 48 and 72 h postinfection and analyzed for PARP and NP by Western blot. The arrows show full-length CCHFV NP (56 kDa), cleaved NP (30 kDa), uncleaved PARP (116 kDa), and cleaved PARP (89 kDa). hr.p.i., hours postinfection. E, MCF-7 and MCF-7-caspase cells were mock-infected or infected with CCHFV (MOI = 1). Mock-infected and CCHFV-infected cells were fixed for 1 h with 4% paraformaldehyde (pH 7.4) at room temperature. Cells were then incubated with permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate. Labeling of DNA strand breaks was performed with the TMR-red cell death detection kit. Values given are the average of three independent experiments. Error bars, S.D. Significance is illustrated with p < 0.05 (*).

FIGURE 7.

Inhibition of or absence of caspase activation increases the yield of progeny virus. SW13-, MCF-7-, and MCF-7-expressing caspase-3 cells were infected with CCHFV (MOI = 1). SW13 cells were mock-treated or treated with Z-DEVD-fmk or Z-FA-fmk. Supernatants of SW13 cells (A) and MCF-7 cells (B) were harvested at 24 and 48 h postinfection (hpi) and analyzed for yield of progeny virus by focus-forming units (FFU). Values given are the average of three independent experiments. Error bars, S.D. Significance is illustrated with p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***).