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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°C but not at 31.8°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.
Aleutian mink disease virus (AMDV) is an autonomous parvovirus which belongs to the genus Amdovirus of the subfamily Parvoviridae (16, 23). Strains of AMDV differ reproducibly in their pathogenicity in animals (26). In adult mink, the virulent AMDV-Utah 1 (AMDV-Utah) strain, which replicates abortively in cell culture, produces a persistent infection associated with severe dysfunction of the immune system (16, 26), including chronic immune complex-mediated glomerulonephritis and arteritis (3, 5, 6, 26, 41). Conversely, the AMDV-G strain, a variant of the AMDV-Utah strain which was adapted to grow permissively in Crandell feline kidney (CrFK) cells at 31.8°C, is nonpathogenic in adult mink (16). The genome of AMDV-G is approximately 97.5% identical to that of the AMDV-Utah strain (13).
The genetic map of AMDV-G has been revised recently. Similarly to the human parvovirus B19 (genus Erythrovirus), all species of AMDV mRNAs are processed from a single pre-mRNA, which is transcribed from the upstream P3 promoter on the 4.7-kb AMDV genome, through alternative splicing as well as alternative polyadenylation (42). The major nonstructural protein, NS1, which is encoded by the R1 mRNA, is essential for viral DNA replication, transcription activation, and capsid assembly (9). The R2 mRNA is the predominant mRNA found throughout productive AMDV infection of the CrFK cells (42, 48). The R2 mRNA contains and expresses the viral capsid proteins VP1 and VP2, as well as the NS2 protein (42). Translation of the capsid proteins is not affected by translation of the NS2 protein, but rather, a cis-acting sequence within the NS2 gene is required for efficient capsid protein production, which has a distinct location dependence (43).
Persistent infections are a feature of many parvoviruses, including minute virus of mice (MVM) (17), human parvovirus B19 (27), and AMDV (15). Control of the synthesis of the capsid proteins, a key feature of any viral infection, has been reported to be the key regulator of B19 tissue tropism (30, 38, 46) and has been shown to govern the ability of AMDV to remain persistent in infected host animals (4, 21, 47, 48). Persistence of AMDV infection in animal systems is characterized by, and likely requires, controlled low levels of capsid protein synthesis.
AMDV is unique among parvoviruses in that infection by AMDV in vivo is restricted (9). However, AMDV infection of CrFK cells induces cell cycle arrest (36) and apoptosis (11). Permissive replication of AMDV in CrFK cells requires activation of caspases (8). Utilizing caspase inhibitors that block the apoptosis dramatically decreases progeny virus production (11). In a recent study, caspases were shown to cleave the large nonstructural protein NS1 at two sites, D227 and D285. This cleavage was required for nuclear localization of NS1 (10). How AMDV infection causes apoptosis in infected cells and undergoes a persistent infection is a central question in AMDV infection.
In this study, we systematically analyzed the proapoptotic nature of AMDV capsid proteins expressed during infection of CrFK cells as well as in transfection of capsid protein-expressing constructs. We found that expression of the capsid proteins by transfection induced apoptosis, represented by active caspases. Strikingly, active caspases cleaved capsid proteins VP1 and VP2, specifically, at amino acid (aa) D420, thereby reducing the number of capsids to encapsidate viral genomes. We found that AMDV-G mutants that are resistant to this cleavage had an increase in progeny virus production at 37°C but not at 31.8°C. Thus, our results may, in part, explain how AMDV infection causes apoptosis in infected cells, reduces capsid protein production and thereafter progeny virus yield, and may initiate persistent infection. Our results identified AMDV capsid proteins VP1 and VP2 as proapoptotic proteins, suggesting that these proteins play an unexpected role in the pathogenesis of AMDV infection.
Crandell feline kidney (CrFK) cells (ATCC CCL-94) were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37°C in 5% CO2. For infection, CrFK cells were infected with AMDV-G at a multiplicity of infection (MOI) of 1 fluorescence focus-forming unit (FFU)/cell (16, 40) and maintained at 31.8°C or 37°C for 6 or 7 days as specified.
AMDV-G was inactivated by UV irradiation as follows: in each well of a 96-well plate, 50 μl of purified AMDV-G was added. The plate was placed in a Hoefer UVC 500 UV cross-linker (Hoefer Inc.) for UV irradiation at a dose of 720 mJ/cm2 (25, 32). To ensure that most of the cells were infected, CrFK cells were infected with the UV-inactivated virus at an MOI of 3 FFU/cell.
For transfection followed by Western blot analysis, CrFK cells were transfected with 1 μg of DNA per well of a six-well plate with Lipofectamine and Plus reagent (Invitrogen) as previously described (31). In some circumstances both in virus infection and in DNA transfection, various inhibitors (caspase inhibitors or proteasome inhibitor) were applied immediately after infection of transfection at concentrations as indicated.
To generate virus from infectious clones, we transfected 2 μg of DNA of pIAMDV or its mutants (described below) into CrFK cells on one 60-mm dish. Cells were maintained at 31.8°C for 7 days and then were lysed by being frozen and thawed three times. Supernatants were collected from each transfection by a brief centrifugation to remove cell debris, one-half of which was used to infect fresh CrFK cells. Reinfected cells then were maintained at 31.8°C for 7 days. Finally, infected cells and media were collected and frozen and thawed three times. Progeny virus was collected by a brief centrifugation of the cell lysates. Virus titers were determined by FFU in CrFK cells at 31.8°C as previously reported (16, 40).
Two general caspase inhibitors, pancaspase fmk inhibitor Z-VAD-fmk (Z-VAD) and an inhibitor with a carboxy-terminal O-phenoxy group (Oph) (Q-VD-Oph [Q-VD]), and nine individual caspase inhibitors (CASP-1, -2, -3/7, -4, -6, -8, -9, -10, and -13 inhibitors) were purchased from R&D Systems (Minneapolis, MN). Proteasome inhibitor MG132 was purchased from Sigma.
All nucleotide numbers for AMDV-G used in this study refer to GenBank accession no. M20036 (13). CMV-NS2mATG-Cap, which has been previously described (43), was renamed for simplicity as plasmid CMV-NS2(−)Cap. CMV-NS2(−)Cap(D407E), CMV-NS2(−)Cap(D413E), and CMV-NS2(−)Cap(D420E) were made by mutating the aspartic acid residues (D) at aa 407, aa 413, and aa 420 to glutamic acid residues (E), respectively, in the VP1/VP2 open reading frame (ORF) of CMV-NS2(−)Cap. CMV-NS2(−)Cap(Utah) was constructed by replacing the AMDV-G VP1/VP2-encoding sequence (nucleotides [nt] 2064 to 4560) with the corresponding sequence from AMDV-Utah (13). CMV-NS2(−)Cap(DVID) was generated by mutating the two leucine (L) residues at aa 418 and 419 to valine (V) and isoleucine (I) residues in the VP1/VP2 ORF of CMV-NS2(−)Cap.
CMV-NS2(−)VP1 and CMV-NS2(−)VP2 were generated by mutating the AUG start codon for VP1 and VP2, respectively, in the CMV-NS2(−)Cap. Mutating both AUG codons for VP1 and VP2 in CMV-HA-NS2-Cap, which has been reported previously (43), resulted in CMV-HA-NS2-Cap(−).
For easy DNA manipulation, we removed the infectious DNA of AMDV-G from pXVB (clone XXI-Q3-15) (12), an infectious clone of AMDV-G containing a pGEM3Z backbone (Promega), to a backbone plasmid derived from pM20, an infectious clone of B19 virus (52). First, a linker of 5′-SalI-GTCGACCCCGCGGGGTACCCGGGCCC-SphI-3′ was inserted into the backbone sequence of SalI/SphI-digested pM20, which resulted in pBBSmaI. Later, pBBSmaI(KOHindIII) was made by blunting the HindIII site. An amplified AMDV-G fragment, SphI-HindIII-nt 1040 to nt 4000-BglII-SalI, was inserted into SphI/SalI-digested pBBSmaI(KOHindIII), which resulted in pBBAMDV-G-nt1040-4000. Then, the DNA fragment from BglII right hairpin end to EcoRI (nt 2554) digested from pXVB was inserted into BglII/EcoRI-digested pBBAMDV-G-nt1040-4000, which made pBBADMVnt1040-RHE. Finally, the fragment from HindIII left hairpin end to SpeI (nt 1083) digested from pXVB was inserted into HindIII/SpeI-digested pBBADMVnt1040-RHE, which produced pBBAMDV-G as a new AMDV-G infectious plasmid. We renamed this plasmid pIAMDV in this study.
All the pIAMDV-based mutants, pIAMDV(D420N), pIAMDV(D420T), pIAMDV(D420H), and pIAMDV(D420E), were made by mutating the aspartic acid (D) at aa 420 to N, T, H, and E residues, respectively. All amino acid numbers for the AMDV-G VP1/VP2 protein used in this study refer to GenBank accession no. AAA66615.
Plasmids pGEX-VP2 and pGEX-VP2(D420E) were constructed by inserting AMDV-G VP2 ORF (nt 2406 to 4347) and mutant VP2(D420E) ORF (with a G at nt 3665) into pGEX4T3 (GE Healthcare). N-terminal glutathione S-transferase (GST) fusion full-length AMDV-G VP2 (GST-VP2) and mutant VP2 [GST-VP2(D420E)] were expressed in Escherichia coli BL21 cells from plasmids pGEX-VP2 and pGEX-VP2(D420E), respectively, according to the manufacturer's protocol (GE Healthcare) by using GST affinity columns attached to the BioLogic LP low-pressure chromatography system (Bio-Rad), as previously described (49).
Western blotting was performed on cell lysates taken 6 days postinfection or 2 days posttransfection as previously described (43). A monoclonal antibody against AMDV-G VP2 aa 428 to 446 (moAb 2126.96.36.199) (14), a rabbit antiserum (R2788) raised against a peptide of aa 428 to 446 on VP2, or a convalescent-phase serum collected from AMDV-infected mink (MAD antiserum) (10), was used to detect AMDV proteins.
Two microliters of the purified proteins (approximately 200 μg/ml) was directly subjected to active caspase cleavage in eluting buffer (phosphate-buffered saline [PBS], pH 7.2, with 10 mM glutathione) with addition of 1 unit of purified active CASP-1, -2, -3, -6, -7, -8, -9, and -10 (Biovision). The mixture was incubated at 37°C for 1 h, resolved by SDS-10% PAGE, and transferred to a nitrocellulose membrane. The membrane then was immunostained with the R2788 antiserum.
The yield of AMDV-G virus physical particles was evaluated by a quantitative real-time PCR assay, which determines genomic copies (gc) of AMDV-G (42). Briefly, approximately 1 × 106 AMDV-G-infected CrFK cells were collected, washed twice with PBS, and resuspended in 5 ml of PBS. Cell suspensions were frozen and thawed three times and centrifuged at 10,000 rpm at 4°C for 10 min. Benzonase (5 μl, 25 U/μl; Novagen) was added to 200 μl of the supernatant, and the solution was incubated at 37°C overnight. Viral DNA was purified by a QIAamp DNA blood minikit (Qiagen) and finally eluted in 200 μl of deionized H2O. The CMVNSCap plasmid was used as a control (1 genomic copy = 5 × 10−12 μg) to establish the standard curve for absolute quantification by using TaqMan technology with an ABI 7500 Fast system (Applied Biosystems, Foster City, CA). The amplicon and the TaqMan minor groove-binding probe were used as previously reported (42). Takara's Premix Ex Taq universal PCR master mix was used for amplification with the standard protocol.
We performed the fluorochrome inhibitor of caspase assay (FLICA) according to the manufacturer's manual (Immunochemistry Tech, Minnesota). Briefly, 1 × 106 live cells were stained with the respective 6-carboxyfluorescein (FAM)-labeled FLICA peptide (FAM-VAD-FMK, FAM-DEVD-FMK, FAM-VEID-FMK, FAM-LEHD-FMK, and FAM-AEVD-FMK for detecting poly-CASP, CASP-3, CASP-6, CASP-9, and CASP-10, respectively). After fixation with 1% paraformaldehyde for 30 min, the cells were permeabilized in 0.3% Tween 20 for 30 min. Rabbit anti-AMDV capsid proteins (R2788) were then used to stain capsid protein-expressing cells at a dilution of 1:100, followed by staining with a Cy5-conjugated anti-rabbit IgG at a dilution of 1:100. Meanwhile, the control groups were stained with antihemagglutinin (anti-HA; Sigma, 1:100 dilution), followed by staining with a Cy5-conjugated anti-mouse IgG at a dilution of 1:100. The cells were then fixed again after antibody incubation and analyzed on the three-laser flow cytometer (LSR II; BD Biosciences) at the Flow Cytometry Core at the University of Kansas Medical Center (KUMC) within 2 h. The Cy5-positive cells were selectively gated and plotted in histogram form to show the FLICA signals of either capsid protein-expressing cells in experimental groups or RFP-HA (HA-tagged red fluorescent protein)-expressing cells and HA-NS2-expressing cells in control groups. All flow cytometry data were analyzed using FACS DIVA software (BD Biosciences).
During AMDV-G infection of CrFK cells or following transfection of either a full-length clone or a minimal construct expressing only capsid genes into CrFK cells, we consistently detected a stable protein product of approximately 26 kDa on Western blots using a monoclonal antibody against an epitope of aa 428 to 446 within the capsid region (moAb 2188.8.131.52) (14). This band was previously designated VPx (42).
To examine whether other cleaved capsid proteins were present during AMDV-G infection, we used a convalescent antiserum (MAD) from an infected mink that can detect nonstructural protein NS1 as well as capsid proteins (10). When lysates of AMDV-G-infected cells were applied, four major bands of AMDV proteins were revealed by the MAD antiserum on SDS-10% PAGE. These bands of VP1, VP2, NS1, and VPx were approximately 78, 71, 70, and 26 kDa in molecular mass (Fig. (Fig.1A,1A, lane 1). The previously identified cleaved NS1 band at approximately 39 kDa was also clearly seen as indicated with an arrowhead in Fig. Fig.1A1A (11). A cleaved band at 50 kDa was lightly seen, as indicated with an arrow in Fig. Fig.1A,1A, and is likely a cleaved VP1 protein. However, a 46-kDa band that corresponds to the residual VP2 fragment that remains after release of VPx was not obviously detected (Fig. (Fig.1A,1A, lane 1), even when a proteasome inhibitor (MG132) was applied to prevent degradation (Fig. (Fig.1A,1A, lane 2).
Next, we transfected a VP1-only-expressing construct [CMV-NS2(−)VP1], a VP1/VP2-expressing construct [CMV-NS2(−)Cap], and a VP2-only-expressing construct [CMV-NS2(−)VP2] into CrFK cells. Blots probed with the MAD antiserum showed that the band of VPx at 26 kDa was obviously detected in cell lysates from all three transfections (Fig. (Fig.1A,1A, lanes 4, 5, and 6, respectively), indicating that the VPx protein can be cleaved from either VP1 or VP2. We observed a significant band at 50 kDa, likely the cleaved VP1 protein (Fig. (Fig.1A,1A, compare lanes 4 and 5 with lanes 6 and 7); however, again, a clear cleaved band at 46 kDa was not detected (Fig. (Fig.1A,1A, lanes 4, 5, and 6). We further used reverse transcription-PCR to probe whether there were additional introns in the capsid-encoding region; however, no intron was identified (data not shown). Thus, we have shown that the capsid proteins VP1 and VP2 of AMDV-G were specially cleaved during infection, which generated a stable cleaved protein, VPx, at 26 kDa. The 46-kDa band that corresponds to the residual VP2 fragment remaining after release of VPx was likely degraded during infection or transfection.
Recently, caspases have been shown to specifically cleave the NS1 protein of AMDV-G during infection (10). We hypothesized that VPx might be generated by caspase cleavage as well. To test this hypothesis, we examined whether VPx cleavage was prevented by caspase inhibitors. In the presence of 50 μM pancaspase inhibitor Z-VAD, the cleaved VPx band was fully abolished when CrFK cells were infected with AMDV-G at 31.8°C (Fig. (Fig.1A,1A, lane 3; Fig. Fig.1B,1B, lane 4). At 37°C, however, 100 μM Z-VAD was required to prevent cleavage during infection (Fig. (Fig.1B,1B, lane 11). In addition, the VPx band was abolished during AMDV-G infection of CrFK cells in a dose-response manner at both 31.8°C and 37°C (Fig. (Fig.1B).1B). These results strongly suggested that this cleavage is mediated through caspase-dependent pathways.
Similarly to our findings with AMDV-G infection, when CrFK cells were transfected with CMV-NS2(−)Cap, the VPx band could be abolished by adding Z-VAD in a dose-response manner at both 31.8°C and 37°C (Fig. (Fig.1C,1C, lanes 1 to 6 and lanes 7 to 12, respectively). Again, the VPx band was not prevented in the presence of the proteasome inhibitor MG132, even at a high concentration of 200 μM (Fig. (Fig.1D),1D), as well as in the presence of a cathepsin inhibitor (data not shown). These results confirmed that the generation of VPx during AMDV-G infection and transfection of the capsid expression construct is due to caspase activity.
Based on the size of the VPx and the epitope (aa 428 to 446) that the moAb 2184.108.40.206 recognizes, we predicted three potential caspase cleavage sites within VP1/VP2, at D407, D413, and D420, respectively (Fig. (Fig.2A).2A). Mutants with mutations of D407E and D413E still generated VPx by transfection (Fig. (Fig.2B,2B, lanes 1 and 2); however, the mutant CMV-NS2(−)Cap(D420E), which contains a mutation of D to E at aa 420, did not produce VPx at all (Fig. (Fig.2B,2B, lane 3). Thus, AMDV-G capsid proteins VP1 and VP2 were specifically cleaved at the site of 417DLLD↓G421, which is a consensus cleavage site of CASP-3/7 (DXXD↓G; “X” indicates any amino acids) (28). This site is on the surface of the AMDV-G capsid according to the structural analysis carried out at 22 Å (33).
To determine which caspase (or pathway) is involved in cleavage of VP1/VP2 at 417DLLD↓G421, we applied individual caspase inhibitors to cells transfected with CMV-NS2(−)Cap. At 50 μM, CASP-6, -8, -9, and -10 inhibitors were more effective at preventing cleavage than CASP-1, -2, -4, and -13 inhibitors (Fig. (Fig.3A)3A) and CASP-3/7 inhibitor (Fig. (Fig.3B).3B). We also used caspase inhibitors from other vendors, e.g., EMD Biosciences (Gibbstown, NJ) and Promega (Madison, WI), and similar results of inhibition were obtained (data not shown). Moreover, an Oph-based pancaspase inhibitor, Q-VD, prevented cleavage of VP1/VP2 effectively at a concentration as low as 5 μM (Fig. (Fig.3C).3C). Q-VD is a recently developed pancaspase inhibitor; it specially inhibits caspase activity and does not cross-inhibit cathepsin (18). Collectively, our results demonstrated that the capsid proteins are specifically cleaved through activation of multiple caspases at the site of 417DLLD↓G421.
To further demonstrate which caspase specifically cleaves VP1/VP2 at 417DLLD↓G421, we applied an in vitro cleavage assay using various active caspases. We expressed a GST-fused VP2 protein. Purified GST-VP2 was then incubated with active CASP-1, -2, -3, -6, -7, -8, -9, or -10. Cleaved bands were detected with the R2788 antiserum. Our results showed that only incubation with active CASP-7 cleaved the GST-VP2 and produced a band at approximately 26 kDa (Fig. (Fig.4A,4A, lane 5). Generation of this cleavage product was largely, although not totally, prevented through a D420E mutation on GST-VP2 (Fig. (Fig.4B,4B, lane 5). This result demonstrated that CASP-7 cleaves GST-VP2 specifically at DLLD↓G in vitro, suggesting that CASP-7 is the effector caspase that cleaves VP1/VP2 in transfection or during infection in vivo.
Infection of AMDV induces a caspase-dependent apoptosis in CrFK cells (11). Since single expression of the capsid proteins by transfection produced the cleaved VPx, which can be prevented by application of caspase inhibitors (Fig. (Fig.11 and and3),3), we hypothesized that expression of AMDV capsid proteins alone can activate caspases. These active caspases, in turn, may contribute to apoptosis during infection. To determine whether caspases are activated and which caspases are most active and to identify the caspase activation pathway triggered by expression of the capsid proteins, we used FLICA to measure activities of both the effector and initiator caspases in either capsid-expressing transfected CrFK cells or AMDV-G-infected CrFK cells.
Expression of capsid proteins following transfection of CMV-NS2(−)Cap significantly induced active caspases in CrFK cells, which were detected by the poly-FLICA assay. We found that approximately 70% of VP1/VP2-expressing cells had active caspases (Fig. (Fig.5A,5A, plot 3) compared with only 27% positive in the control RFP-HA-expressing cells (Fig. (Fig.5A,5A, plot 2). To confirm that activated caspases were induced by expression of the capsid proteins, we transfected CrFK cells with CMV-HA-NS2-Cap, which expressed HA-NS2 and capsid proteins VP1 and VP2 (Fig. (Fig.5C,5C, lanes 1 and 3, respectively), and CMV-HA-NS2-Cap(−), which expressed only HA-NS2 (Fig. (Fig.5C,5C, lanes 2 and 4, respectively). We found that expression of VP1/VP2 by transfection of CMV-HA-NS2-Cap induced a significantly higher level of active caspases than did that of CMV-HA-NS2-Cap(−) in CrFK cells (Fig. (Fig.5A,5A, compare plot 5 with plot 4), suggesting that caspases were activated by expression of the capsid proteins VP1/VP2 rather than merely transfected DNA or VP1/VP2-encoding mRNAs (data not shown). Expression of the HA-NS2 protein did not activate caspases significantly compared to the RFP-HA control (Fig. (Fig.5A,5A, compare plot 4 with plot 2).
We further examined whether capsid proteins of the virulent AMDV strains activated caspases in CrFK cells and whether the caspase cleavage site was conserved in these virulent strains. The capsid proteins of AMDV-Utah contain the same conserved cleavage site (DLLD↓G) as does AMDV-G; nevertheless, we transfected CMV-NS2(−)Cap(Utah) in CrFK cells. Results from Western blot analysis showed that the capsid proteins of AMDV-Utah were cleaved in a similar pattern as those for the AMDV-G capsid proteins (Fig. (Fig.5D,5D, lane 1). Moreover, expression of the AMDV-Utah capsid proteins activated caspases with approximately 82% of the VP1/VP2-expressing cells containing active caspases (Fig. (Fig.5A,5A, plot 6). In addition, we mutated the caspase cleavage site from DLLD↓G to DVID↓G in CMV-NS2(−)Cap. The DVID↓G site is the corresponding putative cleavage site in a virulent AMDV-K strain (35). Again, not only did transfection of CMV-NS2(−)Cap(DVID) significantly induce active caspases (Fig. (Fig.5A,5A, plot 7) but it also produced the cleaved VPx protein (Fig. (Fig.5D,5D, lane 2).
As expected, we detected active caspases in approximately 68% of VP1/VP2-expressing cells during AMDV-G infection (Fig. (Fig.5B,5B, plot 2). In contrast, cells infected with UV-inactivated AMDV-G did not induce active caspases (Fig. (Fig.5B,5B, plot 4), suggesting that capsid entry alone was not sufficient to activate caspases. Inactivation of the UV-AMDV was confirmed by immunofluorescence using anti-NS1 antibody (data not shown).
Collectively, these results demonstrated that caspases are activated extensively during AMDV-G infection as well as in expression of only capsid proteins by transfection, suggesting that AMDV-G capsid proteins are proapoptotic proteins that likely play a key role in inducing apoptosis during AMDV-G infection (10). The cleavage and proapoptotic natures of AMDV capsid proteins are common features of both nonpathogenic and pathogenic AMDV strains.
We performed FLICAs for specific caspases and observed that CASP-10 was the most active caspase in VP1/VP2-transfected cells; 38% of VP1/VP2-expressing cells were CASP-10 FLICA positive. CASP-6, -9, and -3/7 were also more significantly activated in VP1/VP2-expressing cells than in RFP-HA-expressing cells (Fig. (Fig.6A).6A). We observed a similar spectrum pattern of active caspases during AMDV-G infection; 57% and 40% of VP1/VP2-expressing cells were positive for active CASP-10 and CASP-9, respectively (Fig. (Fig.6B).6B). CASP-3/7 and CASP-6 were also active at a percentage of approximately 20% in VP1/VP2-expressing cells during infection (Fig. (Fig.6B).6B). CASP-10 was the most active caspase, not only in transfection but also during infection, further suggesting that CASP-10 is the caspase that initiates the caspase cascade.
Replication of the AMDV genome requires cleavage of the large nonstructural protein NS1 by active caspases, and the use of caspase inhibitors has been demonstrated to decrease virus yield (10). The generation of VPx could significantly reduce the levels of both VP1 and VP2 during infection. Therefore, we cannot simply use caspase inhibitors to determine the biological consequence of the capsid cleavage during AMDV-G infection. Accordingly, we created a series of AMDV-G mutants that contain mutations at the caspase cleavage site from an AMDV-G infectious clone and examined their capability to produce progeny virus.
We obtained four mutants of the infectious clone, pIAMDV(D420N), pIAMDV(D420T), pIAMDV(D420H), and pIAMDV(D420E), which both prevent capsid protein cleavage and maintain the capsid protein as a fully functional capsid for its infectivity, by screening a number of mutants that had mutations at the cleavage site. Transfection of these mutants at 37°C showed that the VPx was significantly abolished (data not shown). We transfected these four mutant clones into CrFK cells, which were maintained at 31.8°C for 7 days. Virus released from cell lysates was used to further infect fresh CrFK cells at 31.8°C for 7 days, as the infectious clone of pIAMDV-G does not produce progeny virus as efficiently as do the infectious DNA clones of other parvoviruses (34, 39, 44, 45, 49). The titers of the final harvested viruses were determined by fluorescence focus-forming units. Then, the same numbers of CrFK cells were infected with these four mutant viruses and AMDV-G at an MOI of 1 FFU/cell either at 31.8°C or at 37°C. Seven days postinfection, yields of progeny virus were quantified as genomic copies (gc) per μl by quantitative real-time PCR. We did not see an increase of progeny virus production from mutants AMDV(D420N), AMDV(D420T), AMDV(D420H), and AMDV(D420E) when cells were cultured at 31.8°C, although they all produced a significant amount of virus (at approximately 1 × 106 to 2 × 106 gc/μl), indicating that they possess a capsid conformation that does not significantly alter their infectivity. However, when infected cells were maintained at 37°C, significant increases of progeny virus from mutants AMDV(D420N), AMDV(D420T), AMDV(D420H), and AMDV(D420E) were observed as shown in Fig. Fig.7A.7A. Furthermore, higher levels of VP1/VP2 expression were detected from cells infected with the mutants (Fig. (Fig.7B).7B). At least an average of three times more virus was produced from infection of the four cleavage-preventing mutants than was produced by the parent virus AMDV-G (Fig. (Fig.7A).7A). Thus, our results suggest that the higher productivity of progeny virus from the cleavage-preventing mutants at 37°C is likely accounted for by the prevention of the caspase cleavage of the capsid proteins.
AMDV is unique among autonomous parvoviruses in that it undergoes persistent infection and thereby causes a chronic disease known as Aleutian mink disease (9). AMDV has been successfully adapted to grow in CrFK cells (36, 40, 41). The initial isolate AMDV-Utah (40) is highly pathogenic to mink, causing persistent infection associated with severe dysfunction of the immune system (16, 26). AMDV-Utah can be propagated through in vivo passages in mink, although in vitro culture in CrFK cells results in only a low titer after multiple serial of passages. In contrast, AMDV-G grows efficiently in vitro in CrFK cells (16) and remains at a higher titer through multiple passages. However, AMDV-G is nonpathogenic to mink and propagates in vivo only at a very low titer (100.5 FFU/ml after 10 mink passages). Interestingly, AMDV-G is a temperature-sensitive mutant that grows at a high titer in CrFK cells only at 31.8°C. The titer of the grown virus at 37°C is at least 10-fold lower than that at 31.8°C (16, 42); the mechanism for this phenomenon is not fully understood. In this study, we demonstrate that expression of AMDV-G capsid proteins activates caspases that, in turn, cleave the capsid proteins at the specific site of 417DLLD↓G421. We also provide evidence that the ability to induce and be cleaved by caspases at the DXXD↓G site is a conserved feature of capsids from AMDV strains, including the pathogenic AMDV-Utah strain and possibly the AMDV-K strain. The caspase-mediated cleavage of capsid proteins reduces progeny virus production significantly at 37°C. This feedback is likely to play a role in maintaining low replication of AMDV and may regulate persistent infection of AMDV in infected mink, since virus replication in mink must take place at 37°C.
It is expected that expression of the AMDV capsid proteins in cell culture results in assembly of empty particles (data not shown and reference 22). The caspase cleavage site (417DLLD↓G421) is located on the surface of AMDV-G capsids (33), suggesting that the cleavage site is accessible on mature capsids and may be targeted. However, it is unknown if capsid cleavage occurs following de novo synthesis of new capsid proteins or following capsid assembly. Furthermore, only a portion of capsid protein is cleaved (approximately 20 to 30% at 31.8°C), implying that either caspase activity or protein cleavage is regulated in some way. To understand the precise role of capsid cleavage in virus replication, future studies are necessary to determine the temporal relationship between capsid protein expression, capsid assembly, and caspase activation.
Specific and precise cleavages of the capsid proteins, or proteolysis in the cytoplasm, have been reported in other parvoviruses. Cathepsin B and L proteins have been shown to bind and cleave intact adeno-associated virus 2 (AAV2) and AAV8 particles in vitro, suggesting that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating (2). AAV2 capsids generate novel fragments that were cleaved at specific sites during purification procedures using trypsin, which is more likely artifactual (50). In full capsids of canine parvovirus (CPV), some VP2 proteins can be converted to the ~63-kDa VP3 by proteolytic cleavage of approximately 19 amino acids from the N terminus (51). In MVM, a third protein, VP3, also is produced after intracellular proteolytic cleavage and removes approximately 25 amino acids from the N terminus of VP2 (24). These specific cleavages of the CPV and MVM capsid proteins are important for intracellular trafficking and infection of the virus. The protease(s) involved in these cleavage events is not yet identified. Previously, it has been reported that AMDV is proteolytically degraded during in vivo infection in mink (1). Three major capsid proteins, at the sizes of 85 kDa, 75kDa, and 27 kDa, in both empty and full particles were detected (1) during in vitro culture. In contrast, only multiple small bands of 30 kDa, 27 kDa, 25 kDa, and 18 kDa were detected from AMDV-Utah propagated from in vivo infection of mink. Our results suggested that this small capsid protein of 27 kDa is likely the VPx in this study and that the degree of capsid cleavage may determine the ability of AMDV to replicate in cell culture. The caspase cleavage site that we identified in this study is conserved in other strains of AMDV (35), suggesting that cleavage of AMDV capsid protein must have a general role in regulating AMDV replication.
The fate of the caspase-cleaved capsid proteins is unknown. A similar phenomenon was observed for NS1 in that not all of NS1 is cleaved by caspases, presumably because there are roles for both full-length and truncated proteins (8, 10). Capsid expression induces active caspases, which in turn cleave NS1 to facilitate DNA replication. Apparently, active caspase is playing roles not only to support AMDV DNA replication but also to cleave capsid proteins, potentially reduce capsid availability, and maintain a persistent infection. However, what prevents caspase-mediated cleavage of viral capsid proteins during de novo synthesis and/or virus assembly is currently unknown.
Apoptosis is defined mechanistically as two pathways involving the sequential activation of caspases. The extrinsic pathway is involved in the binding of ligands (e.g., Fas-L) to the “death” receptors (e.g., Fas) and thereafter in the formation of the DISC (the death-inducing signaling complex) (7), which contains the death receptors, adaptor and initiator CASP-8/10. The intrinsic mitochondrion-mediated (intrinsic) pathway is activated by the multidomain proapoptotic proteins, Bak and Bax (20, 37). In this pathway mitochondrial outer membrane permeabilization (MOMP) is induced, which stimulates the release of cytochrome c into cytosol. The initiator procaspase-9 is thereafter activated within the apoptotic protease-activating factor (Apaf-1) apoptosome complex (29). Activation of CASP-8, -9, and -10 leads to a cascade of activation of downstream effector caspases, including CASP-3, -6, and -7. These caspases, in turn, induce apoptosis (19). Using FLICA, we detected active CASP-10 at a significantly higher level than that of the active CASP-8 and -9 in capsid protein-expressing cells of transfected cells and AMDV-G-infected cells, a finding which suggests that CASP-10 is the initiator caspase. A strong correlation of active caspases between transfection of capsid genes and AMDV-G infection supports the idea that caspase activation induced by the capsid proteins may be the main contributor to activate caspases during infection, albeit in tandem with NS1, which has been reported to induce caspase-dependent apoptosis following transfection into CrFK cells (9). It has been proven that caspase-dependent apoptosis is induced during AMDV-G infection and plays a critical role in viral DNA replication (11). The permissive replication of AMDV-G is caspase dependent and proceeds during virus-induced apoptosis (11). Active caspases cleave the major nonstructural protein NS1 at the sites 224INDT↓S228 and 282DQTD↓S286, which enables the translocation of NS1 from the cytoplasm into the nucleus, where it functions in viral DNA replication (10). CASP-3/7 inhibitors can significantly reduce cleavage of the NS1 (10) and thereafter reduce virus production. Therefore, the effector caspases (i.e., CASP-3, -6, and -7) cleave not only the NS1 (10) but also the capsid proteins.
Generating a mutant virus that both prevents capsid protein cleavage and retains a fully functional capsid for its infectivity is difficult. Fortunately, the DLLD↓G cleavage site is located on the surface of the capsid according to the three-dimensional structure of AMDV-G (33), and thus, we were able to obtain four such mutants that produced significantly more progeny viruses than did the parent virus at 37°C, but not at 31.8°C, suggesting that cleavage of the capsid proteins apparently reduces progeny virus production at 37°C. We consistently observed that less capsid protein, approximately 20 to 30%, was cleaved at 31.8°C, compared to approximately 40 to 50% cleaved at 37°C (Fig. (Fig.1B,1B, compare lane 12 to lane 6). This reduced cleavage is likely due to the lower caspase activity at 31.8°C, a suboptimal temperature. Therefore, our results could support the idea that efficient propagation of AMDV-G in CrFK cells requires an optimal temperature at 31.8°C. We hypothesize that, during in vivo virus infection of infected mink, capsid protein expression activates caspases that efficiently cleave the capsid proteins and the NS1. Cleaved NS1 thus helps to replicate the viral genome. In contrast, cleaved (reduced) capsid proteins limit packaging of the single-stranded viral genome. Thus, a low level of DNA replication of AMDV is balanced during in vivo infection of mink. We conclude that active caspases are critical to replicate AMDV and are likely also important to maintain persistent infection and restriction of virus replication of AMDV in infected mink, in addition to the fact that AMDV is poorly neutralized by antibodies generated during in vivo infection (8, 9).
This work was supported by PHS grant RO1 AI070723 from NIAID and grant P20 RR016443 from the NCRR COBRE program to J.Q. and PHS grants AI46458 and AI56310 from NIAID to D.P. S.M.B. and M.E.B. were supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.
Published ahead of print on 30 December 2009.