The capsid proteins of Aleutian mink disease virus activate caspases and are specifically cleaved during infection

Abstract

Aleutian mink disease virus (AMDV) is currently the only known member of the genus Amdovirus in the family Parvoviridae. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins in vivo. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420 in vitro. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37 degrees C but not at 31.8 degrees C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV.

FIG. 1.

AMDV-G capsid proteins are cleaved by caspases. (A) CrFK cells were either infected with AMDV-G or transfected with VP1/VP2-expressing plasmids as indicated. Infected cells were maintained at 31.8°C with 5% CO₂, while transfected cells were maintained at 37°C with 5% CO₂. At 6 days postinfection or 2 days posttransfection, cells were harvested and lysed in SDS loading buffer. The lysates were subjected to Western blotting using MAD or R2788 antiserum. Detected bands are indicated with their respective designations to the left. Arrow, cleaved VP1 band at 50 kDa; arrowhead, cleaved NS1 band at 39 kDa. (B) CrFK cells were infected with AMDV-G and treated with a pancaspase inhibitor, Z-VAD, at various concentrations as shown. Cells were incubated at either 37°C or 31.8°C as indicated. At 6 days postinfection, cells were harvested and lysed in SDS loading buffer. The lysates were subjected to Western blotting using moAb 282.20.1.4. (C and D) CrFK cells were transfected with CMV-NS2(−)Cap, treated with Z-VAD (C) or proteasome inhibitor MG132 (D) at various concentrations as shown, and maintained at either 37°C or 31.8°C as indicated. At 2 days posttransfection cells were harvested and lysed in SDS loading buffer. The lysates were subjected to Western blotting using moAb 282.20.1.4.

FIG. 2.

AMDV-G capsid proteins are cleaved at the DLLD↓G site. (A) A schematic diagram of the CMV-NS2(−)Cap is shown to the left. Capsid proteins VP1 and VP2 expressed from this construct are diagramed to scale. Putative cleaved proteins are shown with their respective molecular masses in kDa. Predicted cleavage sites targeted by caspase at aa 407, aa 413, and aa 420 are shown with their corresponding mutated glutamic acid residues, which resulted in these three mutants of CMV-NS2(−)Cap as shown in panel B. (B) CrFK cells were transfected with CMV-NS2(−)Cap and the three mutants, as indicated, and maintained at 37°C with 5% CO₂. At 2 days posttransfection, cells were harvested and lysed in SDS loading buffer. The lysates were subjected to Western blotting using moAb 282.20.1.4.

FIG. 3.

Individual and pancaspase inhibitors prevent the cleavage of AMDV-G capsid protein. CrFK cells were transfected with CMV-NS2(−)Cap and treated with individual caspase inhibitors or the pancaspase inhibitor, Q-VD, at various concentrations as indicated. Cells were maintained at 37°C with 5% CO₂. At 2 days posttransfection, cells were harvested and lysed in SDS loading buffer. The lysates were subjected to Western blotting using moAb 282.20.1.4. DMSO, dimethyl sulfoxide.

FIG. 4.

Active CASP-7 cleaves GST-fused VP2 specifically at the site of DLLD↓G. (A and B) Individual active caspases as indicated were incubated with 20 μl of purified GST-VP2 (A) or GST-VP2(D420E) (B). After 1 h of incubation at 37°C, the mixture was resolved by SDS-10% PAGE and immunoblotted with the R2788 antiserum. (C) A schematic diagram of the GST-fused VP2 protein is shown. Cleaved bands and the cleavage site are indicated by arrows. Mutant GST-VP2(D420E) is shown with the mutation of E at aa 420.

FIG. 5.

Caspase activation occurs following expression of AMDV capsid proteins or infection of AMDV-G, but not UV-inactivated virus. (A) CrFK cells were transfected with CMV-NS2(−)Cap, CMV-HA-NS2-Cap(−), CMV-HA-NS2-Cap, CMV-NS2(−)Cap(Utah), CMV-NS2(−)Cap(DVID), and pRFP-HA as a control, respectively. The numbers in plots 1 to 3 show average percentages with deviations of FLICA-positive cells in normal cells (Mock), pRFP-HA-transfected cells (RFP-HA), and AMDV capsid-transfected cells [CMV-NS2(−)Cap]. The numbers in plots 4 to 7 show representative results from at least two independent experiments. (B) CrFK cells were infected with AMDV-G or UV-inactivated AMDV-G (UV-AMDV) as indicated. At 2 days posttransfection at 37°C or at 6 days postinfection at 31.8°C for AMDV-G infection and 2 days postinfection at 31.8°C for UV-AMDV, cells were stained for VP1/VP2 with the R2788 antiserum and for RFP-HA and NS2-HA with an moAb against HA tag (anti-HA; Sigma) and a FLICA peptide bound to all active caspases (poly-FLICA). Stained cells were analyzed by flow cytometry. Mock1 and Mock2 were uninfected CrFK cell controls for AMDV-G and UV-AMDV infections, respectively. The numbers in the plots show representative results (percentages) from at least two independent experiments. (C and D) CrFK cells were transfected with CMV-HA-NS2-Cap, CMV-HA-NS2-Cap(−), CMV-NS2(−)Cap(Utah), and CMV-NS2(−)Cap(DVID). At 2 days posttransfection, cells were harvested and lysed in SDS loading buffer. (C) The lysates from transfection of CMV-HA-NS2-Cap and CMV-HA-NS2-Cap(−) were probed using anti-VP1/VP2 serum R2788 and anti-HA, respectively. (D) The lysates from transfection of CMV-NS2(−)Cap(Utah) and CMV-NS2(−)Cap(DVID) were probed using a convalescent-phase serum (MAD).

FIG. 6.

CASP-10 is the most active caspase in transfection of AMDV-G capsid protein as well as during AMDV-G infection. CrKF cells were transfected with CMV-NS2(−)Cap and pRFP-HA as a control (A) or infected with AMDV-G as indicated (B). At 2 days posttransfection at 37°C or at 6 days postinfection at 31.8°C, cells were stained for VP1/VP2 with the R2788 antiserum and for RFP-HA with an moAb against HA tag (anti-HA; Sigma) and FLICA peptides bound to active individual caspases, CASP-3/7, -6, -8, -9, and -10, as indicated. Stained cells were analyzed by flow cytometry. The numbers in the plots of panel A show average percentages with deviations of FLICA-positive cells in normal cells (Mock), RFP-transfected cells (RFP-HA), and AMDV capsid-transfected cells [CMV-NS2(−)Cap]. The numbers in the plots of panel B show representative results from at least two independent experiments.

FIG. 7.

Prevention of capsid cleavage increases virus production in CrFK cells at 37°C. CrFK cells were infected with progeny viruses from transfection of an infectious clone of AMDV-G and the four mutant viruses as indicated at an MOI of 1 FFU/cell. (A) Seven days postinfection, cell lysates were prepared and digested with benzonase (DNase) overnight. Clarified supernatants that contain DNase-resistant virus particles were used for real-time PCR quantification of virus particles as genomic numbers per microliter (gc/μl). The results show average percentages with deviations from at least three independent infection experiments. The asterisks indicate statistical significance (P < 0.05) by the Student t test, compared to the AMDV-G data. (B) Seven days postinfection, cells were harvested for Western blot analysis with the R2788 antiserum. β-Actin was used as a loading control.