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Journal of Virology, May 2003, p. 5305-5312, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5305-5312.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus

Sonja M. Best, Janie F. Shelton, Justine M. Pompey, James B. Wolfinbarger, and Marshall E. Bloom*

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840

Received 14 November 2002/ Accepted 11 February 2003


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ABSTRACT
 
Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD{downarrow}S) and NS1:285 (DQTD{downarrow}S). Replication of ADV containing either of these mutations was reduced 103- to 104-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.


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INTRODUCTION
 
The struggle for survival between host and virus is a timeless one, often fought at the level of the individual cell. One of the most important cellular defense mechanisms against viral infection is apoptosis. This tightly regulated form of cell death normally functions to remodel tissues during development and to maintain appropriate cell numbers. However, apoptosis following virus infection can limit both the time and the cellular machinery available for replication. Accordingly, many viruses (e.g., adenovirus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, and poxviruses) encode proteins that inhibit apoptosis induction in an effort to prolong cell survival and aid virus replication (15, 19, 20, 29). Viruses can also use apoptosis to their advantage by inducing apoptosis in infected or uninfected cells of the immune system to suppress the immune response (23). Finally, as neighboring cells phagocytose apoptotic cells, viruses can use apoptosis to aid in virus dissemination (35). Hence, there is an intimate and multifaceted relationship between apoptotic pathways, cell death, and viral pathogenesis.

The primary effector proteins of apoptotic pathways are caspases. Following an apoptotic stimulus (e.g., death receptor ligation or oxidative stress), caspases are activated in a cascade and function to cleave and/or activate cytosolic, cytoskeletal, and nucleosomal proteins required for the stepwise dismantling of the apoptotic cell (31, 36, 37). Caspases can also cleave and inactivate essential viral proteins, including the nucleocapsid proteins of influenza and transmissible gastroenteritis corona viruses (14, 39). Caspases are thus one of the specific targets of inhibitory proteins encoded by many different viruses. In sharp contrast to this, we recently showed that Aleutian mink disease parvovirus (ADV) not only induces caspase-dependent apoptosis during permissive replication but also requires caspase activity for efficient virus replication to occur (2). It is unclear how caspases are involved in this novel strategy of caspase-dependent virus replication. However, the requirement for caspase activity raises the possibility that caspases may directly interact with ADV proteins and that this interaction facilitates viral replication.

The coding capacity of ADV and other parvoviruses is limited; the 4.7-kb ADV genome codes for two structural proteins (VP1 and VP2) and two nonstructural proteins (NS1 and NS2). The major nonstructural protein of the autonomous parvoviruses, NS1, is localized predominantly in the nucleus, where it has multiple functions in viral DNA replication, transcriptional regulation, and capsid assembly (9, 10). In addition, NS1 is the primary protein responsible for cytotoxicity to cells (9). Indeed, the NS1 protein of minute virus of mice (MVM), H-1 virus of rats, and B19 virus of humans causes caspase-dependent apoptosis following transfection or infection (8, 24, 26, 30, 34), suggesting that NS1 expression may be directly responsible for the induction of apoptosis. In the present work, we found that caspases cleave ADV NS1 at two sites during virus replication. This cleavage was required for nuclear localization of NS1 and permissive replication of ADV.


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MATERIALS AND METHODS
 
Viruses, cell culture, and baculovirus expression of ADV proteins. A molecularly cloned stock of ADV-G was propagated and assayed in Crandell feline kidney (CrFK) cells as previously reported (3, 4). For infections, 106 CrFK cells were seeded in 12.5-cm2 tissue culture flasks in Dulbecco's minimal essential medium containing 10% fetal calf serum with the specified caspase inhibitor(s) and/or 100 µg of tumor necrosis factor alpha (TNF-{alpha}) (R&D Systems, Minneapolis, Minn.)/ml. After incubation for 24 h at 37°C the cultures were infected with ADV-G at a multiplicity of infection of 10 fluorescent forming units (ffu) per cell. Fresh media containing new inhibitor were added to infected cultures every 12 to 24 h until cells were harvested by scraping after 72 h of incubation at 31.8°C. Cell lysates were prepared by three rounds each of freeze-thawing and 30-s bursts of sonication. ADV-G VP2 particles or NS1 were produced in suspension cultures of Spodoptera frugiperda (Sf9) cells infected with the recombinant baculovirus Autographa californica nuclear polyhedrosis virus encoding either the structural protein of VP2 (6) or full-length NS1 (kind gift of J. Christensen [10, 11]) as previously described.

Antibodies, caspases, and caspase inhibitors. A pool of polyclonal mink anti-ADV (MAD) antisera that recognizes both structural and nonstructural proteins was used for detection of viral proteins by Western blotting (7). Protein A conjugated with horseradish peroxidase was used to detect mink immunoglobulin (Ig) in a Western blot. Rabbit anti-NS1 (residues 173 to 464) antisera and a pool of anti-ADV VP2 monoclonal antibodies were also used in Western blot analysis (5). Additional rabbit anti-NS1 polyclonal antisera used for the immunolocalization of NS1 fusion proteins were R3810 raised against NS1 peptides 2 to 61 and R3125 raised against NS1 peptides 587 to 641. Rhodamine-conjugated goat anti-rabbit IgG (1:10; Roche), fluorescein isothiocyanate-conjugated goat anti-rabbit (1:200; Roche), and fluorescein isothiocyanate-conjugated goat anti-mouse (1:200; Roche) antibodies were used as secondary antibodies in immunofluorescence studies. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) (Roche).

Purified human recombinant caspases 3, 7, and 6 (100 U; 1 U = 1 pmol/min at 30°C) and the inhibitors Ac-DEVD-CHO (caspase 3 inhibitor; 10 µM in cell cultures) and z-VAD-FMK (pan caspase inhibitor; 10 µM in cell cultures) were used (all purchased from Biomol, Plymouth Meeting, Pa.).

Cloning, mutagenesis, and production of NS1 fusion proteins. Full-length ADV-G molecular clones were transformed and amplified in Escherichia coli JC8111 (recBCsbcrecF) by standard techniques. Maintenance of the hairpin terminal repeats was confirmed by restriction enzyme analysis as described previously (17). Other plasmids were maintained in E. coli XL1-Blue (Stratagene, La Jolla, Calif.). The BamHI-NcoI DNA fragment of ADV-G (870 bp) containing the putative caspase cleavage sequences in NS1 was subcloned into Litmus 29 (New England Biolabs, Beverly, Mass.). The residues at positions NS1:212, NS1:227, or NS1:285 were mutated from D to E (NS1:D212E, NS1:D227E, NS1:D285E) by using Quikchange XL site-directed mutagenesis according to the manufacturer's instructions (Stratagene, Cedar Creek, Tex.). The primers used for mutagenesis were as follows with the base change from ADV-G sequence highlighted in bold and the corresponding base numbers in the ADV-G sequence in square parentheses: (NS1:D212E) 5'-CCAGTTCACTTGAGAGAGTATACATTCATATACCTG-3' [824-859]; (NS1:D227E) 5'-GATAAATACAGAGAGTATGGATGG-3' [874-893]; (NS1:D285E) 5'-GTAACTGACCAAACTGAGTCAGCAACCAC-3' [1043-1071]. All mutations were confirmed by DNA sequence analysis. Mutated BamHI-NcoI fragments were then reintroduced into the full-length ADV-G (16).

To confirm that mutated caspase cleavage sequences resisted cleavage by caspases, MBP-NS1173-464 fusion proteins were made. Wild-type and mutated BamHI-NcoI NS1 fragments were ligated into a pMAL expression vector (New England Biolabs) at the C terminus of maltose binding protein (MBP). Following transformation and IPTG (isopropyl-ß-D-thiogalactopyranoside)-induced expression in E. coli(TB1), expression was confirmed by Western blotting with antibodies directed against NS1 and MBP. MBP-NS1 fusion proteins from positive clones were then propagated, purified by affinity chromatography, and concentrated for use in in vitro caspase cleavage studies.

Following confirmation of 224INTD{downarrow}S228 and 282DQTD{downarrow}S286 as the two caspase cleavage sequences present in MBP-NS1173-464, individual 12-cm2 flasks of CrFK cells were transfected by using Effectene (Qiagen) with 1 µg of purified plasmid DNA from clones containing NS1:D227E and NS1:D285E and incubated at 31.8°C for 5 days. Cell pellets were suspended in 1 ml of culture medium, freeze-thawed three times, and sonicated, and the cellular debris were removed by centrifugation, filtered, and passaged into 25-cm2 flasks of CrFK cells. This process was repeated three more times, with the final passage into 150-cm2 flasks. Viruses were titrated in CrFK cells as previously reported (3, 4).

NS1 caspase cleavage products were expressed as fusions with fluorescent proteins to examine their cellular localization. NS11-227 was PCR amplified from full-length NS1 (kindly provided by J. Christensen [9]) and subcloned into pDsRed2-C1 (Clontech, Palo Alto, Calif.) colinear with DsRED (Discosoma sp. red fluorescent protein) by using primers designed to introduce a stop codon following residue 227. NS1286-641 was PCR amplified and subcloned into pEGFP-N3 (Clontech) upstream of the enhanced green fluorescent protein (EGFP) by using primers designed to introduce a methionine preceding residue 286. All sequences were confirmed by DNA sequence analysis, and the constructs were transfected into CrFK cells by using the transfection reagent Effectene.

In vitro cleavage of ADV-G, AcNS1, VP2 particles or MBP-NS1 fusion protein by caspases. ADV-G from infected but untreated CrFK cell cultures, 100 ng of ADV VP2 particles, AcNS1, or MBP-NS1173-464 were incubated either alone or with purified recombinant caspases in a final volume of 20 µl of caspase assay buffer {50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% 3-[(chlolamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 1 mM EDTA, 10% glycerol, 10 mM dithiothreitol} for 2 h at 37°C. Parallel tubes were also incubated with 1 µmol of Ac-DEVD-CHO or z-VAD-FMK. The reaction was terminated by the addition of sample buffer, and proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the anti-VP2 or MAD antibodies.

Cell nuclear extractions and immunofluorescence. For nuclear extraction, CrFK cells were washed three times in cold Dulbecco's phosphate-buffered saline and placed in lysis buffer (40 mM Tri-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.3% Nonidet P-40, 1 mM dithiothreitol) for 10 min on ice. The nuclei were pelleted by centrifugation at 800 x g for 10 min and washed once in lysis buffer without Nonidet P-40 (9). The nuclei and the undiluted supernatant were analyzed by Western blotting. ADV-transfected CrFK cells grown in CC2-treated Lab-Tek chamber slides were fixed in acetone. Immunofluorescent staining for NS1 or capsid proteins was performed as previously described (2).

Western blot analysis. Viral proteins were visualized by Western blotting as previously described (16, 21).


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RESULTS
 
ADV nonstructural protein 1 (NS1) is cleaved by caspases during permissive replication. We have previously shown that efficient permissive replication of ADV requires the activity of specific caspases, including the caspase 3-like caspases (2). The effect of caspases on viral replication may be via direct interactions of caspases with viral proteins resulting in the cleavage of ADV proteins. To test this hypothesis, whole-cell lysates from ADV-infected CrFK cells (72 h postinfection) were incubated with purified caspases 3 or 7. The subsequent generation of viral protein fragments was examined by Western blotting by use of a MAD polyclonal serum that recognizes both structural (the capsid proteins VP1 and VP2) and nonstructural (NS1) proteins (Fig. 1A). Incubation of virus-infected cell lysates with caspases resulted in the production of at least two unique viral protein fragments with apparent molecular masses of 39 and 33 kDa and increased the presence of a third fragment, of approximately 27 kDa. The generation of these fragments was inhibited by the addition of the caspase-3 inhibitor, Ac-DEVD-CHO. Thus, caspases are capable of cleaving ADV proteins.



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  		FIG. 1. Caspases can cleave ADV proteins. (A) ADV-infected CrFK cell lysates were incubated in caspase buffer alone (no treatment) or with caspase 3 (C3) or caspase 7 (C7) ± Ac-DEVD-CHO. Western blots of the lysates were probed with MAD antisera which recognize the two capsid proteins (VP1 and VP2) and the nonstructural protein NS1. Incubation of ADV-infected cell lysates with either caspase 3 or caspase 7 resulted in the appearance of two unique ADV protein fragments (upper two arrows) and the increased presence of a third fragment (lowest arrow). The generation of these fragments was prevented by the addition of caspase inhibitor. (B) ADV-infected cells were untreated or pretreated with z-VAD-FMK, TNF-{alpha}, or TNF-{alpha} + z-VAD-FMK. TNF-{alpha} treatment led to an increased level of ADV NS1 fragments (arrows), the generation of which could be completely prevented by the addition of z-VAD-FMK. (C) Baculovirus-expressed NS1 (AcNS1) was incubated with caspase 3 ± Ac-DEVD-CHO, resulting in its cleavage. (D) ADV VP2 capsids were incubated with caspase 3, 6, or 7 or trypsin and examined by Western blotting using MAD antisera. Caspases were unable to cleave VP2. Caspase 3-mediated cleavage of PARP performed under the same experimental conditions is also shown.

To demonstrate that viral proteins are cleaved by cellular caspases during permissive replication of ADV, CrFK cells were treated 24 h prior to infection with the apoptotic stimulus TNF-{alpha}, which can sensitize cells to apoptosis via caspase-dependent mechanisms. Cells were also treated prior to infection with z-VAD-FMK or with TNF-{alpha} plus z-VAD-FMK (Fig. 1B). The low-molecular-mass bands of 39 and 27 kDa were present in untreated ADV-infected cells. Pretreatment of infected cells with z-VAD-FMK prevented the appearance of these bands. In contrast, pretreatment of cells with TNF-{alpha} resulted in an increase in the level of these products compared to that of untreated ADV-infected cells, as well as the appearance of the 33-kDa fragment. The addition of z-VAD-FMK at the same time as TNF-{alpha} again inhibited the appearance of these cleavage products. Cell lysates from TNF-{alpha}-treated uninfected cells did not react with the MAD antisera (not shown). Importantly, these results confirm that caspase-mediated cleavage of viral proteins occurs during permissive replication of ADV in CrFK cells.

To determine if caspases were cleaving ADV structural and/or nonstructural proteins, purified NS1 (AcNS1) (Fig. 1C) or VP2-only capsids (Fig. 1D) expressed by recombinant baculoviruses were incubated with purified caspases. Incubation of PARP [poly(ADP-ribose) polymerase] with caspase 3 was included as a positive control for caspase 3 activity (Fig. 1D). Caspases 7 and 6 were also active under these experimental conditions as shown by their ability to cleave NS1 (data not shown). Caspases cleaved AcNS1 (Fig. 1C) at two sites generating NS1 cleavage products of approximately 46, 39, and 33 kDa. However, caspases failed to cleave VP2 (Fig. 1D). This identified NS1 as the target of caspase activity in these assays.

Identification of caspase cleavage sites in NS1. Examination of the ADV-G NS1 amino acid sequence revealed two putative caspase cleavage sequences, 224INTDS228 and 282DQTDS286. Cleavage at NS1:D227 and NS1:D285 would produce products similar in size to those observed in infected cells. To determine if these putative caspase recognition sequences were bona fide cleavage sites, we mutated the aspartic acid (D) residues to glutamic acid (E) residues (32) at each site and introduced the mutations into MBP-NS1173-464 (with an approximate molecular mass of 75 kDa, approximately 42 kDa of which is MBP). A third mutant was made at NS1:D212 as a control due to its resemblance to a caspase cleavage site and its proximity to NS1:D227. These mutant MBP-NS1 fusion proteins were purified and incubated with purified caspase 3 in vitro (Fig. 2A). Cleavage of MBP-NS1:D212E occurred at two sites similar to those of the wild-type protein generating two predominant products of 58 and 49 kDa, suggesting that D212 was not involved in a caspase cleavage site. However, only the 58- or 49-kDa proteins were produced following incubation of MBP-NS1:D227E or MBP-NS1:D285E with caspase 3, respectively. As only a portion of NS1 that spans the two cleavage sites was used in this MBP fusion protein (NS1:173-464), the sizes of the cleavage products are not the same as in cells. Therefore, the fragments generated after in vitro caspase cleavage are not directly comparable to those found in ADV-infected cells. The absence of a second cut site in these two mutant fusion proteins demonstrates that these two sequences are indeed the sites at which caspases cleave NS1 (Fig. 2B).



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  		FIG. 2. Identification of 224INTDS228 and 282DQTDS286 as caspase cleavage sequences in NS1. (A) MBP-NS1173-464 fusion proteins in which single amino acid changes were made (D to E at NS1:D212E, NS1:D227E, or NS1:D285E) were produced and then incubated with caspase 3 (C3) ± Ac-DEVD-CHO (Inh.). Mutations at D227 or D285 prevented cleavage of NS1 at those two positions, thus identifying INTDS and DQTDS as caspase cleavage sequences. (B) Schematic diagram showing the two caspase cleavage sites in NS1, the resulting cleavage products, and their predicted molecular masses (m.w.). (C) Schematic diagram indicating functional domains known for ADV NS1 (9) and by extrapolation from MVM NS1.

Mutation of caspase cleavage sites in full-length ADV clones. We next determined the effect of the D-to-E mutations on the viability of full-length clones of ADV-G. Following confirmation of the caspase sequences, the NS1:D227E and NS1:D285E mutations were cloned into an infectious clone of ADV-G, which was then transfected into CrFK cells. A double mutant was also made (NS1:D227E/D285E). The changes made in these viruses do not alter other open reading frames, nor are they proximal to splice donor or splice acceptor sites for any of the ADV mRNA splices. It is therefore unlikely that any effects observed would be due to direct effects on nonstructural proteins other than NS1. Infectious virus from the transfections was passaged a further three times, after which the titer of wild-type ADV was maximal at approximately 107 ffu/ml (Table 1). Replication of both single mutants NS1:D227E and NS1:D285E was restricted approximately 1,000- to 10,000-fold compared to that of the wild-type virus (Table 1). Three clones of the double mutant were tested, and none of these were replication competent. However, viral proteins were observed by immunofluorescence assay in CrFK cells following the initial transfection (not shown), demonstrating that transfected plasmid DNA entered the cells and directed the synthesis of viral proteins.


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  		TABLE 1. Replication of ADV containing mutations in caspase cleavage sites

Immunofluorescent staining of viral capsid and NS1 protein (using antisera against NS1173-464) following primary transfection with either of the two single mutants yielded two interesting observations (Fig. 3). First, NS1 from viruses containing NS1:D227E or NS1:D285E localized almost exclusively to the cytoplasm in every cell (Fig. 3B), whereas wild-type NS1 protein was localized to the nucleus (Fig. 3A). Secondly, capsid VP2 protein expression was not detected in cells in which NS1 was in the cytoplasm (Fig. 3B). Therefore, in the absence of appropriate caspase cleavage, NS1 is improperly localized and capsid protein expression is highly restricted. Staining of viral proteins in the fourth-round passage of the single mutants yielded similar results to that of the transfections, although approximately 50% of NS1-positive cells demonstrated both cytoplasmic and nuclear localization of NS1, while the remaining 50% showed only cytoplasmic localization (data not shown). Although capsid protein was not readily observed by IFA during infection with the NS1 single mutants, some must be produced to contribute to the production of progeny virus. We do not believe that nuclear localization of mutant NS1 after four passages represents NS1 expression from reverted viruses, since subsequent passages of NS1:D227E and NS1:D285E viruses did not result in increased virus titers (data not shown). These results suggest that the presence of one caspase cleavage site is sufficient for some nuclear localization of NS1 and subsequent virus replication, although both sites are required for these processes to be at their most efficient.



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  		FIG. 3. Immunofluorescent localization of capsid and NS1 proteins of wild-type and caspase cleavage mutant ADV clones. (A) Following transfection of wild-type ADV, both NS1 (red) and VP2 capsid (green) proteins were observed in the nucleus. (B) Following transfection of the NS1 caspase cleavage mutant, NS1:D285E, NS1 localized to the cytoplasm and no capsid proteins were observed.

NS1 caspase cleavage fragments are differentially localized in the cell. To investigate how cleavage of NS1 may affect its nuclear localization, we determined the cellular localization of the NS1 cleavage products. Nuclear and cytoplasmic extractions were made from ADV-infected cells (either untreated or pretreated with z-VAD-FMK) and analyzed by Western blotting using MAD antisera (Fig. 4). The majority of full-length NS1 was present in the nucleus. The 46-kDa and 39-kDa C-terminal NS1 cleavage fragments were also predominantly localized to the nucleus. The N-terminal 33-kDa fragment was not detected in either fraction. The 27-kDa fragment was present in both nuclear and cytoplasmic fractions. The band below the 27-kDa band in Fig. 4 may also be the hypothesized 6.5-kDa NS1 cleavage product (Fig. 2B) that is present in the cytoplasmic fraction, although this fragment was never well resolved. Pretreatment of cells with z-VAD-FMK resulted in lower overall levels of ADV protein production, consistent with our previous report (2), and no NS1 cleavage products were detected in either cellular compartment. These results suggest that following cleavage by caspases the C-terminal portions of NS1 become localized to the nucleus whereas the N-terminal portions are both cytoplasmic and nuclear in distribution.



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  		FIG. 4. Differential localization of NS1 caspase cleavage products in the nuclei and cytoplasm of ADV-infected CrFK cells. Whole-cell (W), nuclear (N), and cytoplasmic (C) fractions of uninfected or ADV-infected ± z-VAD-FMK CrFK cells show the localization of the four main NS1 caspase cleavage products. The C-terminal fragments with apparent molecular masses of 46 kDa (i) and 39 kDa (ii) are predominantly localized to the nucleus, whereas the N-terminal fragment of 27 kDa (iv) was evenly distributed in both the nucleus and the cytoplasm. The N-terminal 33-kDa fragment (iii) was not detected in either fraction.

To confirm the differential cellular localization of NS1 fragments, the N-terminal (residues 1 to 227) caspase-cleaved fragment of NS1 was fused to the C terminus of dsRED and the C-terminal (residues 286 to 641) fragment of NS1 was fused to the N terminus of EGFP and expressed in CrFK cells (Fig. 5). EGFP:NS1286-641 localized almost exclusively to the nucleus as shown by both EGFP localization (green) and the immunolocalization of NS1 using a polyclonal antiserum raised against NS1:587-641 (red). This was despite the high molecular mass of EGFP:NS1286-641 (64 kDa), suggesting that there may be an intact nuclear localization sequence within residues 286 to 641 of NS1. EGFP alone, dsRED alone, or dsRED:NS11-227 localized uniformly across both the nucleus and cytoplasm of transfected cells. In the case of dsRED:NS11-227, expression and localization of NS1:1-227 were confirmed by immunolocalization of NS1 with a polyclonal antiserum raised against NS1:2-61 (shown in green). The cellular localization of NS1:1-227 and NS1:286-641 by immunofluorescence assay is consistent with the Western blot analysis of cell fractions shown in Fig. 4. Taken together, these results suggest that the C-terminal caspase cleavage fragments are actively transported to the nucleus following transfection or infection.



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  		FIG. 5. NS1:286-641 localizes to the nucleus of CrFK cells by 24 h posttransfection. Expression of the reporter protein (EGFP or dsRED), immunolocalization of the NS1 portion of the fusion protein, DAPI-stained nuclei, and the merged image following transfection of pEGFP, pEGFP:NS1286-641, pDsRED, or dsRED:NS11-227 are shown. EGFP:NS1286-641 almost completely localizes to the cell nucleus, whereas dsRED:NS11-227 is more uniformly distributed between the nucleus and the cytoplasm.


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DISCUSSION
 
Permissive replication of ADV is caspase dependent and proceeds during virus-induced apoptosis (2). In the present paper we have shown that caspases cleave the major ADV nonstructural protein, NS1, at two sites (224INTD{downarrow}S228 and 282DQTD{downarrow}S286) and that this cleavage enables the translocation of NS1 from the cytoplasm to the nucleus. Furthermore, mutation of both cleavage sites rendered the virus replication defective while ablation of either site independently partially inhibited replication of the virus in cell culture. Thus, cleavage of NS1 at one of the two sites is likely to be sufficient for some nuclear localization and function of NS1.

These findings immediately suggest a mechanism for caspase-dependent ADV replication. Parvoviral NS1 performs several obligatory roles in virus replication that require NS1 translocation into the nucleus (28, 38). Since caspase cleavage appeared to be necessary for ADV NS1 to locate in the nucleus, permissive replication can proceed only if caspase cleavage occurs. In MVM, an NLS facilitates this translocation (25). Therefore, caspase-mediated cleavage of NS1 in ADV replication may expose an as-yet-unidentified NLS or remove a cytoplasmic retention sequence. Either mechanism might allow NS1 to localize to the nucleus. Evidence for such a regulatory mechanism for NS1 translocation in parvoviruses already exists. Although NS1 is predominantly a nuclear protein, 30 to 40% of wild-type MVM NS1 is retained in the cytoplasm of infected cells (25). However, both a naturally occurring 65-kDa NS1 derivative (NS1*) that lacks approximately 18 kDa of the C-terminal sequence (12) and a mutant of NS1 (dlC67) that lacks the C-terminal 67 residues (25) localize almost exclusively in the nucleus. These observations suggest that the C-terminal portion of MVM NS1 contains a cytoplasmic retention sequence. It is not known how MVM NS1* is generated or if its presence is required for MVM replication. However, as this protein is disproportionately represented in the nucleus, it is tempting to speculate that it is important and that caspase cleavage of NS1 may supply an equivalent protein during ADV replication.

Despite the facts that purified NS1 was efficiently cleaved by caspases in vitro and that caspase cleavage is necessary for permissive replication, much of the NS1 in infected cells and nuclei was full-length. In addition, the NS1 cleavage fragments were differentially distributed in the cell, with the C-terminal fragments present in the nucleus (Fig. 4). These findings suggest that the role of caspase cleavage of NS1 in replication may be complex. The multiple replicative functions of NS1 (or Rep proteins) require the formation of an NS1 homodimer and its movement into the nucleus (13, 25, 27, 33). Because caspase cleavage of ADV NS1 was also required for nuclear localization of full-length protein, it is possible that the truncated C-terminal portion of NS1 facilitates the nuclear translocation of full-length NS1. For this to be a viable hypothesis, the truncated NS1 must retain a number of features including the ability to translocate to the nucleus and to interact with full-length NS1. This could occur in several ways. First, the truncated form may simply act as a chaperone for full-length NS1. Alternatively, an oligomer formed between full-length and truncated NS1 molecules might be functional. Finally, the processing of NS1 may have other, less obvious roles in virus replication or cytotoxicity.

These findings raise interesting questions regarding the role of ADV NS1 in virus replication. Several functional domains have been mapped to NS1 (Fig. 2C). For caspase-cleaved NS1 to be functional in an NS1 oligomer, the truncated form of NS1 must be functional in DNA binding capacity, ATPase, and transcriptional activation. The binding of ATP by NS1 ATPase is a major event that precedes oligomerization, and oligomerization itself is required for specific binding of NS1 to parvovirus DNA and transactivation (9, 10). Caspase-mediated cleavage of ADV NS1 at D285 generates a C-terminal fragment that contains the intact ATPase/helicase as well as a putative transcriptional activating domain. In addition, this fragment contains a region of charged amino acid residues in a coiled-coil-like arrangement (NS1 residues 538 to 593) that has been shown to be important in the oligomerization of parvovirus nonstructural proteins. It is therefore possible that the C-terminal fragment acts as more than a chaperone of full-length NS1 and may have active roles in viral replication.

Caspase-mediated regulation of replication provides a possible mechanism for ADV persistence. The targets for ADV persistent infection in adult mink are lymph node macrophages, in which the replication is noncytopathic and restricted (1, 22). Due to their pivotal role in coordinating immune responses, apoptosis induction in macrophages is tightly controlled, and caspase activation following infection with ADV may be limited. We demonstrated here that limitation of caspase activity and apoptosis following ADV infection may constrain fully permissive replication, and this may allow viral persistence at a cellular level. As this work is the first description of caspase-dependent parvovirus replication, it will be important to determine if this is a feature of other autonomous parvoviruses that typically demonstrate permissive replication. However, mink enteritis virus does not require caspase activity for replication in the same CrFK cell line (2), and thus our findings may be unique to ADV. The direct regulation of virus replication by caspases is decidedly unusual and may be an important and novel mechanism that contributes to the replication strategies of other lytic viruses that establish noncytolytic, persistent infections in vivo.

The caspase recognition site in NS1 at 282DQTDS286 is conserved among all isolates of ADV studied to date, although the 224INTDS228 motif is present only in ADV-G, ADV-SL3, and ADV-Utah (18). This conservation implies that caspase activity and NS1 cleavage are important in the replication of both pathogenic and nonpathogenic viruses and supports the idea that caspase activity could regulate virus replication in animals as well as in cell culture. Experiments are in progress to investigate the precise role of NS1 cleavage in caspase-dependent replication of ADV both in cell culture and in adult mink. We now have an example of a virus that, instead of inhibiting the function of caspases, utilizes caspase activity to directly facilitate virus replication. These findings add another intriguing dimension to the relationship between virus infection and apoptosis, which may prove to be important in the replication strategies of other persistently infecting viruses.


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FOOTNOTES
 
* Corresponding author. Mailing address: Rocky Mountain Laboratories, 903 S. Fourth St., Hamilton, MT 59840. Phone: (406) 363-9275. Fax: (406) 363-9286. E-mail: mbloom{at}niaid.nih.gov. Back


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Journal of Virology, May 2003, p. 5305-5312, Vol. 77, No. 9
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.9.5305-5312.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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