Functional and biochemical characterization of the baculovirus caspase inhibitor mavip35

Functional and biochemical characterization of the baculovirus caspase inhibitor mavip35


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ABSTRACT Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of


caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, _Autographa californica_ (Ac) P35 and _Spodoptera littoralis_ (Spli) P49 have


been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its ‘reactive site loop’ then becomes


trapped, irreversibly bound to the cleaved inhibitor. The _Maruca vitrata_ multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found


that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct


from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 _in vitro_, but (in contrast to


AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive


to cleavage by DRONC _in vitro_, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC _in vitro_. SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL BASIS FOR THE


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US11 Article Open access 19 July 2022 MAIN Cell death is essential for normal animal development and to destroy pre-cancerous and auto-immune cells, but it has been postulated that apoptosis


originally evolved to defend primitive multicellular organisms against intracellular pathogens such as viruses.1 Over evolutionary time, an ‘arms race’ has developed between viruses and


their hosts. Cellular machineries detect infection and activate self-destruction pathways, limiting the ability of the virus to replicate and spread to other cells. Viruses, in turn, have


evolved ways to suppress their hosts’ apoptotic machineries during the early phase of infection. Targeting caspases is one approach adopted by viruses to block their host cells’ suicidal


reaction to infection.2 The P35 family is a group of caspase inhibitors encoded by viruses that infect insects. Almost all of the viruses that possess P35 relatives are baculoviruses:3 the


sole exception known to date is the _Amsacta moorei_ entomopoxvirus.4 No cellular P35 homologs have been described as yet, although as baculoviruses usually derive their genes from their


hosts,5 it seems likely that P35 genes did evolve from a cellular ancestor. The best-studied P35 family member is AcP35, encoded by the baculovirus _Autographa californica_ multi


nucleopolyhedrovirus (AcMNPV).6 It inhibits caspases via a substrate trap mechanism.7, 8, 9 The caspase cleaves AcP35 within the reactive site loop. This cleavage provokes a conformational


change within the inhibitor, targeting its amino terminus to the caspase's active site, preventing hydrolysis of a thioester adduct between the inhibitor and the protease, and thus


locking the caspase in an inactive, P35-bound form.7 Of the many mammalian, insect and nematode caspases tested, very few were found to be insensitive to AcP35. The _Drosophila_ initiator


caspase DRONC was shown to be resistant to inhibition by AcP35.10, 11 Processing of downstream _Spodoptera_ caspases proceeded in the presence of AcP35,12 implying that a _Spodoptera_ DRONC


ortholog (denoted ‘Sf-caspase-X’) is also resistant to AcP35 inhibition. AcP35 could inhibit the enzymatic activity of recombinant caspase 9 (DRONC's mammalian counterpart), however


extremely high concentrations of AcP35 were required to prevent apoptosome-activated caspase 9 from cleaving its physiological substrate, caspase 3.13 This suggests that AcP35 cannot


efficiently interfere with the function of naturally activated caspase 9. _Bombyx mori_ nucleopolyhedrovirus (BmNPV) encodes a protein (BmP35), which shares 91% of its amino-acid sequence


with AcP35. BmP35 displayed only weak anti-apoptotic activity14 and, unlike AcP35, BmP35 was dispensable for normal viral propagation.15, 16 Extracts from mammalian cells expressing BmP35


were less potent than lysates from AcP35-expressing cells at inhibiting recombinant caspase 3, although lower BmP35 expression levels may have contributed to this difference.13 No


quantitative data have been published regarding the caspase inhibitory potency or specificity of BmP35, and no other close relatives of AcP35 have been functionally or biochemically


investigated to date. Some baculoviruses encode distant relatives of AcP35, which constitute the P49 subfamily. _Spodoptera littoralis_ (Spli) NPV-P49 is the best-studied member of this


subfamily. Like AcP35, SpliP49 is a broad-spectrum caspase inhibitor that could suppress insect17, 18, 19, 20 and mammalian21 cell death. Unlike AcP35, SpliP49 could inhibit DRONC-mediated


yeast lethality,21 but it was incapable of preventing DRICE processing in _Drosophila_ cells.19 SpliP49 could, however, prevent processing of executioner _Spodoptera_ caspases,18, 20


implying that it can inhibit the proposed Sf-caspase-X. AcP35 contains the cleavage sequence DQMD’G within its reactive site loop, but SpliP49 instead possesses the sequence TVTD’G at this


position. This sequence is required for SpliP49 to inhibit the distal insect caspase Sf-caspase-X, but its insertion into the AcP35 reactive site loop failed to confer this capability,20


indicating that other regions of the SpliP49 protein, not shared by AcP35, are critical for its ability to inhibit insect initiator caspases. The caspase inhibitor AMVP33 from _Amsacta


moorei_ entomopoxvirus is the least homologous member of the P35 superfamily, exhibiting only 25% amino acid identity to AcP35.4 The baculovirus _Maruca vitrata_ (_Mavi_) MNPV infects the


legume pod borer _Maruca vitrata_, and may offer a biological means of controlling this important pest of legume crops.22 The recent sequencing of the _Mavi_MNPV genome23 revealed the


presence of a P35 ortholog (MaviP35). The predicted MaviP35 protein is highly homologous to AcP35, but its predicted reactive site loop possesses a distinct caspase cleavage sequence. Here,


we report our characterization of the apoptosis and caspase inhibitory properties of MaviP35. RESULTS Sequencing of the _Mavi_MNPV genome23 revealed that this virus encoded a P35 ortholog


that was 81% identical to the founding member of this family, AcP35 (Figures 1a and b), and modeling suggested the two relatives may adopt similar structures (Figures 1c and d). Residues


determined to be essential for the caspase inhibitory activities of AcP35, including C28 and D87,24, 25 were conserved in MaviP35, suggesting it too may function as a caspase inhibitor that


could prevent apoptosis. We tested this hypothesis by overexpressing MaviP35 in mammalian and insect cells, and monitoring the transfectants’ sensitivity to apoptosis. MaviP35 inhibited


insect cell death and caspase activity triggered by infection with an AcP35-deficient baculovirus, although less efficiently than AcP35 (Figures 2a and b). MaviP35 and AcP35 inhibited insect


cell death induced by actinomycin D to a similar extent (Figure 2c), but MaviP35 protected a larger proportion of insect cells than AcP35 against apoptosis induced by UV irradiation (Figure


2d). MaviP35 and AcP35 afforded similar levels of protection to mammalian cells against cisplatin-induced apoptosis (Figure 2e), but MaviP35 was less protective than AcP35 against death


induced by TNF-related apoptosis-inducing ligand (TRAIL; Figure 2f). We have previously exploited yeast-based assays to visualize caspase activity and inhibition, and these were used to


provide an indication of the specificity of MaviP35 for various caspases. MaviP35 protected yeast from death induced by mammalian caspases 1, 2, 3, 5, 7 and 8, the _Drosophila_ caspases


DCP-1 and DRICE, and CED-3 from _Caenorhabditis elegans_ (Figure 3). In this system, MaviP35 appeared to exhibit similar activity to AcP35, and protected yeast from death induced by caspases


5, 8 and CED-3 better than SpliP49 (Figure 3). AcP35 has been shown to inhibit caspases via a pseudosubstrate mechanism, and mutation of the caspase cleavage site abolishes caspase and


apoptosis inhibitory activity.7, 8 Comparison of the MaviP35 and AcP35 sequences predicted that caspases may cleave MaviP35 after residue D87 within the site TQFD87′G (Figure 1a). Consistent


with this residue being critical for caspase inhibition, a putative P1 mutant failed to prevent DRICE induced yeast death (Figure 4). Lysates from yeast expressing active DRICE exhibited


considerable DEVDase activity. Co-expression of untagged or carboxyl terminally FLAG-tagged wild-type MaviP35 or AcP35 abolished this activity, but their cleavage site mutants had negligible


impact (Figure 4). Immunoblotting confirmed that the tagged mutants were expressed at least as abundantly as their wild-type counterparts. Interestingly, cleavage products could not be


detected in lysates from yeast co-expressing DRICE with either MaviP35-FLAG or AcP35-FLAG. Because DRICE activity in this system is generated through autoactivation, we suspect that only


relatively few P35 molecules would be required to block this feedback loop, and immunoblotting may not be sufficiently sensitive to detect this small number of cleaved proteins. The yeast


system is a sensitive tool for observing caspase inhibition within a naive eukaryotic environment, but only provides limited insight into the strength of inhibition. To gain a quantitative


understanding of MaviP35's caspase inhibitory activity, we purified FLAG-tagged MaviP35 and AcP35 and examined their ability to prevent recombinant caspases from cleaving fluorogenic


substrates _in vitro_. When present at 100–1000-fold excess, MaviP35 diminished by at least 90% the activity of caspases 2, 3, DRICE, DCP-1 and CED-3. Weaker inhibition was seen for caspases


1 and 7. MaviP35 only partially reduced the activity of caspases 6, 8 and 9, and negligible inhibition of caspase 10 was observed (Figure 5). A particularly striking difference between


AcP35 and MaviP35 related to inhibition of caspase 8. As observed previously,26 AcP35 potently suppressed the proteolytic activity of this enzyme, yet a 100-fold excess of MaviP35 only


decreased its activity by about half (Figure 5). The alignment of P35 subfamily members revealed that the P4 and P2 residues differed between AcP35 and MaviP35 (P4-DQMD-P1 _versus_


P4-TQFD-P1, respectively). Mutagenesis studies of AcP35 had previously demonstrated that changing its P4 aspartate residue to either alanine or asparagine markedly impaired its ability to


inhibit caspases 3 and 8,7 highlighting the importance of the P4 amino acid for caspase inhibition. The cleavage site of MaviP35, containing a P4 threonine residue, was reminiscent of the


site at which DRONC auto-processes between its large and small subunit (TQTE)11 and, to a lesser extent, the caspase cleavage site within the SpliP49 reactive site loop (TVTD).17 This


prompted us to wonder whether MaviP35 may be the first example of a P35 subfamily member that can inhibit DRONC. Consistent with this notion, expression of MaviP35 completely abolished


DRONC-mediated yeast death (Figure 6a) and recombinant DRONC could cleave purified MaviP35, although not as efficiently as DRICE (Figures 6b and c). We therefore sought to determine whether


MaviP35 could suppress DRONC activity in insect cells. DRONC can cleave DRICE between its large and small subunits10, 11 and cleavage of DRICE in _Drosophila_ cells has previously been used


as a readout of DRONC activity.19, 27 A GFP-tagged active site mutant of DRICE expressed in _Drosophila_ Kc167 cells was completely processed to yield a 39-kDa product following actinomycin


D treatment (Figure 6d), as expected from DRONC cleavage between the large and small subunits of DRICE (Figure 6e). Enforced expression of DIAP1 completely inhibited cleavage of


DRICEC211A-eGFP in actinomycin D-treated _Drosophila_ cells (Figure 6). Curiously, AcP35 partially inhibited DRICEC211A-eGFP cleavage, with more than half of the protein remaining intact in


cells co-expressing AcP35. MaviP35 also partially inhibited this cleavage event, although less potently than AcP35. Purified MaviP35 did not impair the ability of recombinant DRONC to cleave


a peptide substrate (Figure 6f). Using a range of substrate and inhibitor concentrations, inhibition by bacterially produced DRONC of MaviP35 was extremely weak (Figure 7). Quantitation of


MaviP35's inhibition of other caspases confirmed the data shown in Figure 5: strong inhibition of executioner caspases, but weak to negligible inhibition of initiator caspases (Figure


7c). DISCUSSION This study describes the apoptosis and caspase inhibitory properties of a new P35 subfamily member: MaviP35. Like AcP35, MaviP35 could inhibit insect and mammalian cell


death, was susceptible to caspase cleavage, and could inhibit the proteolytic activity of caspases. Nevertheless, AcP35 and MaviP35 differed in their specificity profile. MaviP35 inhibited


executioner apoptotic caspases with similar potency to AcP35. However, MaviP35 was substantially less potent than AcP35 at inhibiting mammalian caspases 8 and 10. This may explain the weaker


protection afforded by MaviP35 relative to AcP35 against TRAIL-induced cell death. MaviP35 was also a weaker inhibitor of recombinant caspase 9 than AcP35. However, it is important to note


that published data suggest that the susceptibility of recombinant caspase 9 to AcP35 _in vitro_ is not mirrored by apoptosome-activated caspase 9 within cell lysates,13 so it is possible


that AcP35 and MaviP35 are both incapable of interfering with the activity of naturally activated caspase 9 _in vivo_. The P4 residue of MaviP35 (threonine) differs from that of AcP35


(aspartate). Mutation of P4 in AcP35 to asparagine reduced its ability to inhibit caspase 3 by 47-fold,7 yet MaviP35 – which contains the slightly larger polar uncharged residue threonine at


this position – inhibited caspase 3 with similar efficiency to wild-type AcP35. Presumably differences in other regions of the protein compensate, allowing MaviP35 to efficiently suppress


caspase 3 activity. The MaviP35 cleavage site resembles the auto-processing site between the large and small subunits of DRONC, and MaviP35 was sensitive to DRONC proteolysis _in vitro_.


MaviP35 inhibited yeast death triggered by high-level expression of DRONC, suggesting that it could function as a pseudo-substrate inhibitor of DRONC. However, two pieces of evidence argue


against this possibility. First, MaviP35 was an extremely weak inhibitor of recombinant DRONC activity _in vitro_. Its _K_i was indistinguishable from that of AcP35, which was incapable of


inhibiting DRONC in yeast and _in vivo_10, 11 and was resistant to DRONC cleavage _in vitro_. Second, MaviP35 only partially inhibited actinomycin D-induced cleavage of the DRICEC211A–eGFP


fusion protein, impeding processing to a lesser extent than AcP35. This result implies that actinomycin D treatment provoked DRONC-mediated (AcP35/MaviP35 resistant) processing of the


DRICE-eGFP substrate in Kc167 cells, but also suggested that other proteases, sensitive to AcP35/MaviP35 inhibition, also contributed to this proteolysis. Taken together, these data lead us


to postulate that MaviP35 functions as a classical substrate for DRONC, rather than as a suicide substrate inhibitor. It is possible that the ability of MaviP35 to suppress DRONC-mediated


yeast lethality reflects substrate competition: DRONC's proteolytic attention may be diverted to MaviP35 processing rather than cleavage of essential yeast proteins. Despite the high


homology between MaviP35 and AcP35, this study has revealed an important difference in their caspase specificity. Both inhibitors could suppress downstream caspases from insects and mammals


(and CED-3), but only AcP35 could efficiently block the activities of mammalian caspases-8 and -10. Neither AcP35 nor MaviP35 could significantly inhibit the _Drosophila_ initiator caspase


DRONC. MATERIALS AND METHODS SEQUENCE COMPARISONS AND STRUCTURAL MODELING Sequences of the P35 subfamily members were aligned using the ‘Multialign’ program28 and formatted using Jalview


2.0.29 Genbank accession numbers for the P35 sequences used were: _Mavi_MNPV: YP_950833, _Ac_MNPV: NP_054165.1, _Ro_MNPV: NP_703122, _Bm_NPV: AAO12972, _Hycu_NPV: AAO17287, S_plt_NPV:


CAA71304 and _Lese_NPV: AAF78504. Protein Homology/analogY Recognition Engine (Phyre) was used to predict the structure of MaviP35.30 AcP35 and predicted MaviP35 structures were depicted


using Jmol: an open-source Java viewer for chemical structures in 3D (http://www.jmol.org/). PLASMID CONSTRUCTION For yeast experiments, coding DNA sequences were expressed from inducible


Gal1/10 promoters.31 Plasmids-expressing caspase 2, caspase 3-lacZ, caspase 5, caspase 753, caspase 8, CED-3, reverse DRICE, DCP-1, DRONC, AcP35, AcP35-F, AcP35D87A-F and SpliP49-F have been


described previously.11, 21, 32, 33, 34 Other plasmids were generated as follows: the caspase 1 coding region was amplified using oligonucleotides 1 and 2 from pET21b-Casp-1-His (purchased


from Addgene Cambridge, MA, USA). The product was cut with _Bam_HI/_Xba_I and ligated into pGALL-(_LEU2_)32 cut with _Bam_HI/_Xba_I. The _MaviP35_ coding region was amplified with


oligonucleotides 3 and 4 from a plasmid kindly donated by Prof. Chung-Hsiung Wang, cut with _Bam_HI/_Xho_I and ligated into pGALL-(_HIS3_)32 cut with _Bam_HI/_Xho_I. _MaviP35_ was also


amplified with oligonucleotides 3 and 5 and cloned the same way into pGALL-(_HIS3_), to incorporate a carboxyl terminal FLAG tag. Site-directed mutagenesis to generate _MaviP35__D87A_ was


performed using PCR. After excising _MaviP35_ from pGALL-(_HIS3_)-MaviP35 and inserting it into Bluescript II SK+ via _Bam_HI/_Xho_I, a PCR was conducted using _MaviP35-F_ as a template


(oligonucleotides 6 and 7) to amplify the 3′ portion of the gene incorporating a P1 mutation. This product was cut with _BclI_/_Xho_I and inserted into _BclI_/_Xho_I cut MaviP35-Bluescript


II SK+. The resulting P1-mutated _MaviP35-F_ gene was then excised with _Bam_HI/_Xho_I and ligated into pGALL-(_HIS3_). For mammalian and insect cell expression, _MaviP35-F_ was amplified


from pGALL-(_HIS3_)-MaviP35-F with oligonucleotides 8 and 7, then cut with _Bam_HI/_Xho_I, blunted and inserted into pHSP70PLVI+CAT (chloramphenicol acetyl transferase)35 cut with


_BglII_/_EcoR_I blunted or pEF36 cut with _Bam_HI/_Xba_I blunted. _Caspase 3_ was amplified using primers 9 and 10, then digested with _Nde_I/_Xho_I and ligated into _NdeI_/_Xho_I cut


pET23a. All amplified fragments were sequenced to verify the absence of any unintentional mutations. The pAct5eGFP vector was kindly provided by Samuel Le Fort and David Vaux. The coding


sequencing of _DRICE__C211A_ was amplified from pGMR-DRICEC211A (described below) using primers 11 and 12, cut with _BglII_ and cloned into pACT5eGFP cut with _Bam_HI to generate


pACT5c-DRICEC211A-eGFP. Restriction analyses and sequencing were performed to determine the orientation of the insert and to check the sequence was correct. pGMR-DRICE was made by amplifying


_DRICE_ with primers 13 and 14, then cutting the product with _Bgl_II and _Xba_I and cloning into pGMR.37 pGMR-DRICEC211A was constructed by amplifying the 3′ portion of _DRICE_ encoding


the active site with a mutagenic forward primer (15) and wild-type reverse primer (16), digesting the product with _Nhe_I and _Not_I and ligating into pGMR-DRICE cut with _Nhe_I and _Not_I.


The DNA encoding eGFP was removed from pAct5eGFP by cutting with _Bam_HI and _Xba_I, blunting using Klenow polymerase and religating to yield pAct5c. _Bam_HI/_Xba_I fragments encoding either


DIAP1 (obtained by digesting pGALL-(_HIS3_)-DIAP1) or MaviP35-FLAG (excised from pGALL-(_HIS3_)-MaviP35-F) were ligated into _Bam_HI/_Xba_I cut pAct5c-eGFP, replacing the _eGFP_ gene. The


sequences of the nucleotides referred to above were as follows: * 1:: 5′-CGGGATCCATGGCCGACAAGGTCCTGAAGGAG-3′ * 2:: 5′-GCTCTAGATTAATGTCCTGGGAAGAGGTAGAAACATC-3′ * 3::


5′-GGGATCCCATATGTGTGTAATTTTTCCAGTAG-3′ * 4:: 5′-GCCTCGAGTTAATCAATGTTTAATATTATATTG-3′ * 5:: 5′-GCCTCGAGTTACTTGTCATCGTCGTCCTTGTAGTCCATATCAATGTTTAATATTA TATTGTTG-3′ * 6::


5′-CAATTTGATCAACTAGAACGCGACCACAGCACTCAATTCGCT GGAGGCC-3′ * 7:: 5′-CTTTATTATTTTTATTTTATTGAGAGGGTGG-3′ * 8:: 5′-GCGGATCCGCCATGTGTGTAATTTTTCCAGTAG-3′ * 9::


5′-GGAATTCCATATGGAGAACACTGAAAACTCAGTGG-3′ * 10:: 5′-CCCTCGAGGTGATAAAAATAGAGTTCTTTTGTGAGC-3′ * 11:: 5′-GTCAGATCTCAAAATGGACGCCACTAACAATGGAG-3′ * 12:: 5′-GTCAGATCTACCCGTCCGGCTGGAGCCAAC-3′ *


13:: 5′-CGAGATCTCCGCCATGGACGCCACTAACAATGGAGAATCC-3′ * 14:: 5′-CGTCTAGACTAAACCCGTCCGGCTGGAGCCAACTGC-3′ * 15:: 5′-CCTCGCTAGCCGGCAAACCCAAGTTGTTCTTCATACAGGCCGCCCAGGGC-3′ * 16::


5′-GCACTAGTGCGGCCGCCTAAACCCGTCCGGCTGGAGCCAACTGC-3′ APOPTOSIS ASSAYS FROM INSECT CELLS Sf21 cells were plated at 8 × 105 cells per well in six-well plates in TC-100 insect medium (Invitrogen,


Carlsbad, CA, USA) plus 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA, USA), and allowed to attach overnight at 27°C. Transfections were performed using lipofectin, which


was prepared as a 1.5 : 1 mixture of DOTAP ((_N_-(1-(2,3-Dioleoyloxy)propyl)-_N_,_N_,_N_,-trimethylammonium chloride salt; Avanti Polar Lipids, Alabaster, AL, USA) and DOPE (L-α


Phosphatidylethanolamine, dioleoyl; Sigma-Aldrich, St. Louis, MO, USA). For each well, 2.5 _μ_g of the eGFP expression plasmid pHSP70GFPBsu36I38 was mixed with 2.5 _μ_g of either


pHSP70PLVI+CAT, pHSP70PLVI+AcP35 or pHSP70PLVI+MaviP35F. This DNA was diluted to 100 _μ_l with TC-100 lacking FBS and incubated for 5 min at room temperature (RT). In a separate tube, 6 _μ_l


lipofectin was diluted to 100 _μ_l with TC-100 lacking FBS and incubated for 5 min at RT. The mixtures of DNA and lipofectin were then combined and allowed to incubate for 15 min at RT.


During this incubation the Sf21 cells were washed twice with 1 ml of TC-100 lacking FBS. After the last wash, 800 _μ_l of TC-100 lacking FBS was left in each well and the DNA/lipofectin


mixture (200 _μ_l) was added to each well and allowed to incubate at 27°C for 4 h. The mixture plus the media were removed and 2 ml of TC-100 plus 10% FBS was added to each well. The cells


were heat shocked 24 h post transfection at 42°C for 30 min, to drive expression from the hsp70 promoter. The cells were then induced to undergo apoptosis 4 h post heat shock, by treatment


with either UV (by placing the plates on a transilluminator for 10 min) or actinomycin D (Invitrogen; 250 ng/ml). To determine cell viability, the number of GFP-expressing cells was counted


in each well both immediately before and 17 h after UV or actinomycin D treatment. Three random fields of view per sample were counted per well, and three separate wells were assayed per


treatment. To assay sensitivity to infection-mediated apoptosis, Sf9 cells (106) were plated in six-well culture dishes for 2 h in the TC-100 medium with 10% FBS. After 2 h, the medium was


replaced with Grace's insect unsupplemented medium (Invitrogen). Cells were transiently transfected with pHSP70PLVI+CAT, pHSP70PLVI+AcP35 or pHSP70PLVI+MaviP35F using 3 _μ_g of each


plasmid and 6 _μ_l of lipofectin. Transfection mixtures were replaced with TC-100 plus 10% FBS after 5 h incubation with cells. Cells were infected at 24 h post transfection with


vAcP35KO-PG39 at a multiplicity of infection of 1 PFU/cell, and then harvested at 48 h post infection for caspase and viability assays. Caspase assays were performed using the substrate


Ac-DEVD-AFC (MP Biomedicals, Solon, OH, USA) as described previously.40 To assess viability, three random fields of view were photographed ( × 200 magnification), and viable cells were


counted for each well. Cell viability was determined by counting the non-apoptotic cells and comparing to the number of viable cells in a mock-infected control at 0 h post infection, which


was set at 100%. DRICE CLEAVAGE ASSAYS IN KC167 CELLS Two million Kc167 cells (kindly provided by Gary Hime) were transfected with 0.2 _μ_g of either pAct5c-eGFP or pAct5c-DRICE-eGFP plus


1.8 _μ_g of either pAct5c, pAct5c-MaviP35-F or pAct5c-DIAP1, using the Effectene transfection reagent (Qiagen, Doncaster, Victoria, Australia) according to the manufacturer's


instructions. After 24 h transfection, the cells were incubated in media containing 0 or 1 _μ_M of actinomycin D for 12 h. Cells were lysed in mammalian lysis buffer (50 mM Tris pH 7.5, 375 


mM NaCl, 1 mM ethylenediamine tetra-acetic acid, 1% Triton X-100) containing protease inhibitors (protease inhibitor cocktail set 1; Calbiochem, Darmstadt, Germany) and subjected to 12%


SDS-PAGE and either Coomassie stained or immunoblotted using anti-GFP (Roche Applied Science no. 11814460001; Castle Hill, New South Wales, Australia) or anti-FLAGM2 (Sigma no. F3165) and


anti-mouse IgG-HRP (Sigma no. A9044). MAMMALIAN APOPTOSIS ASSAYS SV-40 transformed mouse embryonic fibroblasts (MEFs) were co-transfected with 1 _μ_g of CMV-lacZ and either 3 _μ_g of pEF,


AcP35-pEF or MaviP35-pEF using FuGENE HD transfection reagent (Roche; Basel, Switzerland). LN18 glioblastoma cells (ATCC; Manassas, VA, USA) were co-transfected with 1 _μ_g of CMV-lacZ and


either 2 _μ_g of pEF, AcP35-pEF or MaviP35-pEF using Lipofectamine (Invitrogen). Transfections were performed according to the manufacturers’ instructions. Twenty-four hours after


transfection, the medium was removed and the cells were incubated with fresh unsupplemented media or media containing cisplatin (Mayne Pharma, Mulgrave, Victoria, Australia) or Superkiller


(crosslinked) TRAIL (Alexis Biochemicals, Lausen, Switzerland). After 24 h, the cells were stained with 5-bromo-4-chloro-3-indolyl-_β_-D-galactopyranoside and the blue cells were scored for


viable _versus_ apoptotic morphology, as previously published.36 YEAST TRANSFORMATION AND DEATH ASSAYS _Saccharomyces cerevisiae_ yeast strain W303_α_ was transformed31 and analyzed in


survival assays21 as described previously.31 Caspase 1 was expressed using the pGALL-(LEU2)-Casp1 vector described above. PROTEIN ASSAYS FROM YEAST Yeast transformants were grown and


transgene expression induced as described previously.11 The lysates were subjected to SDS-PAGE and the gels were then stained with Coomassie brilliant blue (Sigma) to visualize protein


loading and immunoblotted as described previously.11 The membranes were probed with an antibody recognizing the FLAG tag (clone M2; Sigma) and anti-mouse-HRP (Sigma). To measure the caspase


activity via fluorescence analysis, the yeast were treated as follows: An overnight culture was pelleted, washed twice with 1 ml of TE (Tris HCl 10 mM pH 8, EDTA 1 mM) and induced for 6.5 h


in complete media containing 2% galactose. After pelleting the yeast culture, the yeast was weighed and glass beads were added. To lyse the cells, 5 ml of CelLyticY reagent (Sigma) with 10 


mM DTT was added per 1 g of yeast cells. After gently shaking the cells for 30 min at RT, the debris was removed by centrifugation at 16 100 × _g_ for 10 min at 4°C. The protein


concentration of the supernatant was measured using the Bicinchoninic acid protein assay kit (Sigma). In fluorescence assays, lysate (0.5 mg/ml) was mixed with Ac-DEVD-AFC (100 _μ_M) in


DRICE activity buffer (50 mM HEPES pH7.5, 10% sucrose, 0.1% CHAPS, 5 mM DTT, 100 mM NaCl, 1 mM EDTA), and the fluorescence (excitation 410 nm, emission 500 nm) was measured every 30 s for 1 


h. The slope of each curve was calculated using Graphpad Prism 5.0 (La Jolla, CA, USA). The slope of each curve determines the concentration of free AFC per minute, which provides a measure


of caspase activity. PROTEIN PURIFICATION FROM YEAST Transformants were grown in 5 ml glucose-containing selective medium to stationary phase, then expanded by addition of 195 ml


glucose-containing selective medium. After expanding the cells for 16–18 h, the pelleted yeast were washed once with 100 ml TE and resuspended in 1 l of galactose-containing complete media


for 6.5 h induction. Cells were harvested at 12 100 × _g_, 4°C for 15 min then glass beads were added after weighing the pellets. To lyse the cells CelLyticY reagent (Sigma) was used as


described above. The supernatant was incubated for 30 min at 4°C with 200 _μ_l Anti-FLAG M2 affinity gel (Sigma), which had been previously washed three times with 12 × resin volume washing


buffer (Tris HCl 50 mM pH 7.4, 150 mM NaCl). Incubated beads were pelleted 5 min at 3750 g, RT then washed with 32 resin volumes of washing buffer for 10 min at 4°C. Five elution fractions


were collected each using one resin volume of elution buffer (1 mM HEPES pH 7.0, 0.1% PEG, 0.001% CHAPS, 0.1 mM DTT, 200 ng/_μ_l FLAG peptide (Sigma)). For each elution step the beads were


incubated for 2 min at 4°C with agitation, and supernatant was collected after a pelleting for 1 min at 16 100 g, 4°C. After subsequent SDS-PAGE analysis and Coomassie brilliant blue (Sigma)


staining, the fractions containing pure FLAG-tagged proteins were pooled, and protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad, Gladesville, New South Wales,


Australia). RECOMBINANT CASPASES Caspases 1, 2, 6, 7, 8, 9 and 10 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). BL21-(DE3)-pLysS (Merck, Darmstadt, Germany) bacteria were


transformed with caspase 3-pET23a (described above) or the following previously published plasmids: DCP-1-pET23a,32 DRICE-pET23a,32 CED-3-pET23a,34 DRONC-pET23a.11 Caspase 3, DCP-1, DRICE


and CED-3 were purified as described previously.34 DRONC purification was carried out as follows: A transformant colony was inoculated into 1.5 ml 2YT-amp/chlor (16 g/l tryptone, 10 g/l


yeast extract, 5 g/l NaCl, 100 _μ_g/ml ampicillin and 35 _μ_g/ml chloramphenicol) and grown overnight at 37°C. A volume of 1 ml of this culture was expanded into 50 ml of pre-warmed


2YT-amp/chlor and grown at 37°C for 2 h, 200 r.p.m. A total of 10 ml of this was mixed with 190 ml of pre-warmed 2YT amp/chlor in a 2-l baffled flask and shaken at 37°, 200 r.p.m. until


OD600 reached 0.6–0.8. IPTG was added to a final concentration of 1 mM and the culture was shaken at 20°C for 18 h, then pelleted for 10 min at 3000 g, 4°C and then frozen at −80°C. The


pellet was thawed then resuspended in a 10-ml Bug Buster Mastermix (Merck) by pipetting, then incubated for 20 min at RT. Insoluble cell debris was removed by centrifugation at 16 100 × _g_


for 20 min at 4°C. Half a milliliter of NiNTA resin (Qiagen) was washed twice in phosphate buffer (50 mM NaHPO4, 300 mM NaCl), then incubated with the induced bacterial lysate for 30 min at


4°C, gently mixing. The beads were washed twice with phosphate buffer containing 5 mM imidazole, then the caspase was eluted with phosphate buffer containing 250 mM imidazole. _IN VITRO_


QUANTITATION OF CASPASE INHIBITION Caspases 1, 2, 3, 6, 7, 8, 9, 10, DRICE, DCP-1 and CED-3 were pre-activated for 10 min at 37°C in universal caspase citrate buffer (10 mM HEPES pH 7.0, 10%


sucrose, 0.1% CHAPS, 10 mM DTT, 100 mM NaCl, 1 mM EDTA, 0.65 M Na-Citrate). After the activation step, the caspase was incubated either with buffer alone, F-CED-91–251, AcP35-F or with


MaviP35-F for 1 h at 37°C. The appropriate fluorescent substrate was then added (100 _μ_M): Ac-WEHD-AFC for caspase 1; Ac-VDVAD-AFC for caspase 2; Ac-DEVD-AFC for caspases 3, 7, DRICE, DCP-1


and CED-3; Ac-VEID-AFC for caspase 6; Ac-LEHD-AFC for caspases 8, 9 and 10 and Ac-TQTD-AFC for DRONC (Enzo Life Sciences). Fluorescence (excitation 410 nm, emission 500 nm) was measured


every minute for 2 h. The maximal slope of each curve was calculated using Prism 5.0 and graphed. DETERMINATION OF INHIBITION CONSTANTS Caspases were pre-activated for 10 min at 37°C in the


following buffers: caspase 3: 100 mM HEPES pH 7.0, 10% PEG, 0.1% CHAPS, 10 mM DTT; DRICE: 50 mM HEPES pH 7.5, 10% sucrose, 0.1% CHAPS, 5 mM DTT, 100 mM NaCl, 1 mM EDTA; caspases 8 and 9: 10 


mM HEPES pH 7.0, 10% sucrose, 0.1% CHAPS, 10 mM DTT, 100 mM NaCl, 0.1 mM EDTA, 0.65 M Na-Citrate; DRONC: 50 mM Tris pH 7.4, 100 mM NaCl, 0.65 M Na-Citrate. Subsequently, the caspase was


incubated with either AcP35-F or MaviP35-F in the appropriate activity buffer for 1 h at 37°C. Substrates were added at concentrations ranging from 0.001 to 1000 _μ_M. Substrates used were:


Ac-DEVD-AFC for caspase 3 and DRICE; Ac-LEHD-AFC for caspases 8 and 9 and Ac-VEID-AFC for DRONC (Enzo Life Sciences). Fluorescence (excitation 410 nm, emission 500 nm) was measured every


minute for 2 h. The slope of each curve was calculated using Prism 5.0. Inhibition constants were calculated by non-linear regression using Prism 5.0 software, using a competitive inhibition


model as described by these equations: _K_mObs=_K_m × (1+[_I_]/_K_i) and _Y_=_V_max × _X_/(_K_mObs+_X_), where [_I_] is the inhibitor concentration (_μ_M); _K_i is the inhibition constant


(_μ_M), _V_max is the maximum enzyme velocity (relative fluorescence units (RFU)/min), _K_m is the Michaelis–Menten constant (_μ_M), _X_ is the concentration of substrate (_μ_M) and _Y_ is


the change in fluorescence (RFU/min). ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * AAF78504 * AAO12972 * AAO17287 * CAA71304 * NP_054165.1 * NP_703122 * YP_950833 ABBREVIATIONS * Mavi:


_Maruca vitrata_ * MNPV: multiple nucleopolyhedrovirus * Ac: _Autographa californica_ * Bm: _Bombyx mori_ * NPV: nucleopolyhedrovirus * Spli: _Spodoptera littoralis_ * TRAIL: TNF-related


apoptosis-inducing ligand * FBS: fetal bovine serum * MEF: mouse embryonic fibroblast * Xgal: 5-bromo-4-chloro-3-indolyl-_β_-D-galactopyranoside * CAT: chloramphenicol acetyl transferase *


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replication in mosquito cells. _J Gen Virol_ 2008; 89: 2651–2661. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Chung-Hsiung Wang for providing the


plasmid bearing the _MaviP35_ gene, Sam Le Fort and David Vaux for the pAct5c-eGFP plasmid, Gary Hime for the Kc167 cells and Anissa Jabbour and Paul Ekert for the MEF cells. This work was


funded by the National Health and Medical Research Council project grant to CJH and RJC (#602525), an Australian Research Council Future Fellowship to CJH (#FT0991464), scholarships to ILB


and MML from the La Trobe University and a scholarship to ILB from the Cooperative Research Centre for Biomarker Translation. AUTHOR INFORMATION Author notes * M M Green, S Civciristov and D


Pantaki-Eimany: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University,


Bundoora, Victoria, Australia, I L Brand, M M Green, S Civciristov, D Pantaki-Eimany, C George & C J Hawkins * Division of Biology, Kansas State University, Manhattan, KS, USA T R Gort, 


N Huang & R J Clem * Children's Cancer Centre, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia C J Hawkins Authors * I L Brand View


author publications You can also search for this author inPubMed Google Scholar * M M Green View author publications You can also search for this author inPubMed Google Scholar * S


Civciristov View author publications You can also search for this author inPubMed Google Scholar * D Pantaki-Eimany View author publications You can also search for this author inPubMed 


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inPubMed Google Scholar * N Huang View author publications You can also search for this author inPubMed Google Scholar * R J Clem View author publications You can also search for this author


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DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by P Salomoni RIGHTS AND PERMISSIONS This work is licensed under the Creative


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THIS ARTICLE CITE THIS ARTICLE Brand, I., Green, M., Civciristov, S. _et al._ Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35. _Cell Death Dis_ 2,


e242 (2011). https://doi.org/10.1038/cddis.2011.127 Download citation * Received: 01 November 2011 * Accepted: 14 November 2011 * Published: 15 December 2011 * Issue Date: December 2011 *


DOI: https://doi.org/10.1038/cddis.2011.127 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not


currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * caspase * P35 * baculovirus * apoptosis * infection *


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