Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) do not inhibit caspase-3 and caspase-7 processing in cells exposed to pro-apoptotic inducing stimuli

We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.     

Sequential Activation of Caspases and Serine Proteases (Serpases) During Apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TLCK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP- reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.

Key Words:

Serpases, Caspases, Apoptosis, Enzymatic center, Chymotrypsin, FLICA, FLISP, Cell necrobiology     

Protease Inhibitors Cause Necrotic Cell Death in Chlamydomonas reinhardtii by Inducing the Generation of Reactive Oxygen Species

Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N‐tosyl‐lysine‐chloromethyl‐ketone (TLCK) and N‐tosyl‐phenylalanine‐chloromethyl‐ketone (TPCK), which affect the trypsine‐ and chymotrypsine‐like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.     

Effects of proteinase inhibitors on fertilization in sea lamprey (Petromyzon marinus)

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.     

Sequential activation of caspases and serine proteases (serpases) during apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.     

The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations In Vitro

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.     

Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases.

Bcl-2 family proteins and interleukin-1-beta converting enzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basic regulators of apoptosis. However, the precise mechanism by which they interact is unclear. In this study, we found that gamma-radiation-induced apoptosis of leukemia cells was associated with activation of multiple caspases and bax up-regulation. Membrane changes and caspase activities were suppressed by specific caspase inhibitors. Similarly, the serine protease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also prevented caspase activation and poly(ADP-ribose) polymerase cleavage in vivo but had no effect on caspase activity in vitro. TLCK also prevented bax up-regulation as a result of its inhibitory effect on p53 function. Inhibitors of caspases and serine proteases partially prevented cell death, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signaling events in Bax-mediated cell death, including steps upstream and downstream of p53 and bax up-regulation.     

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

Interaction of protease inhibitors with the catalytic subunit of cAMP-dependent protein kinase

The inactivation of the catalytic subunit from rabbit muscle cAMP-dependent protein kinase by the chloromethyl ketones from lysine and phenylalanine (TLCK and TPCK; A. Kupfer et al. (1979) Proc. Natl. Acad. Sci. USA $\text{76}$, 3073) has been confirmed for the same enzyme from rat muscle. However, other structurally not related protease inhibitors, antipain and leupeptin, did not inhibit the catalytic subunit from rat muscle. Thus it seems to be critical to attribute the interference of protease inhibitors with complex biological phenomena like tumorigenesis etc. generally to the inhibition of protein kinases.     

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP.1 cells differently dependent on both the concentration used and the apoptotic stimulus. TPCK (50 - 75 microM) induced both biochemical and ultrastructural changes characteristic of apoptosis, including proteolysis of poly (ADP-ribose) polymerase (PARP) and lamins together with formation of large kilobase pair fragments of DNA, particularly of 30 - 50 and 200 - 300 kilobase pairs in length but without internucleosomal cleavage of DNA. The induction of apoptosis by TPCK also involved the processing of CPP32 and Mch 3 to their catalytically active subunits. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE-like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concentrations of TPCK (5 - 20 microM) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 microM) inhibited cell death induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell survival, the second in facilitating internucleosomal cleavage of DNA and the third in the modulation of apoptosis induced by different apoptotic stimuli.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. 5. Role of proteases, inhibitors, and substrates

The effect of protease inhibitors and substrates on the killing of tumor cells by in vitro activated macrophages was examined. Phenylmethylsulfonyl fluoride, a serine esterase inhibitor, and the active site titrants TPCK and TLCK (chloromethyl ketone derivatives of tosyl-l-phenylalanine and tosyl-l-lysine) inhibited macrophage-mediated cytotoxicity in a dose-dependent and irreversible fashion. The synthetic protease substrates, tosyl-lysine methyl ester and tosyl-arginine methyl ester and the natural esterase inhibitors soybean trypsin inhibitor and antithrombin III had no effect. These results suggest that an activated macrophage-associated esterase may play a modulating role in the cytolytic interaction between activated macrophages and tumor cells.     

Suppression of phorbol ester-enhanced radiation-induced malignancy in vitro by protease inhibitors is independent of protein kinase C

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.     

Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.     

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.     

The Characterization of Cell Death Induced by 1-(3-C-Ethynyl-β-D-ribo-Pentofuranosyl)Cytosine (ECyd) in FM3A Cells

Abstract

The characterization of cell death induced by 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine(ECyd), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells in vitro. Accompanied with the cell death induced by ECyd (3.0 μM)-treatment, about 100–200 kbp-sized and internucleosomal DNA fragmentation were observed by orthogonal-field-alternation gel electrophoresis (OFAGE) and conventional gel electrophoresis, respectively. Protease inhibitors, carbobenzoxy-L-aspart-1-yl[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH₂-DCB), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), effectively blocked the cell death, suggesting that the proteases inhibited by Z-Asp-CH₂-DCB, TLCK or TPCK were involved in the process of the cell death.     

A chymotryptic-type protease inhibitor decreases interleukin 2 synthesis and induces prostaglandin production in Jurkat T cells

TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethylketone), a potent inhibitor of chymotryptic-type serine proteases, was found to decrease IL2 synthesis in Jurkat T cells. Conversely, the tryptic-type protease inhibitor, TLCK (N-alpha-p-tosyl-lysine chloromethylketone), which structurally is very similar to TPCK, had no effect on IL2 synthesis. Prostaglandin synthesis, a process that is known to reduce IL2 production in T cells, was increased by TPCK but not by TLCK, suggesting that this process could be, at least in part, responsible for the inhibition of IL2 production. Our results imply that a chymotryptic-type serine protease plays an active role in the regulation of IL2 synthesis and thus in the whole process of T-lymphocyte activation.     

Reduction in cell entry of Eimeria tenella (Coccidia) sporozoites by protease inhibitors, and partial characterization of proteolytic activity associated with intact sporozoites and merozoites

The role of proteases in the invasion of host cells by Eimeria tenella (Wisconsin strain) was studied in vitro. Protease inhibitors were used to treat sporozoites before inoculation or were applied to cultured chicken kidney cells before infection. The inhibitors antipain, leupeptin, aprotinin, L-1-tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), or N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced parasite invasion to 16-66% of control after treatment of cultured cells or sporozoites with 5- or 50-micrograms/ml concentrations of inhibitors in the culture medium. Phenylmethylsulfonyl fluoride (PMSF) reduced invasion to 32-57.7% at concentrations of 1-4 mM. The optimum pH for hydrolysis of azocasein by intact sporozoites or merozoites was determined over a range of pH 5.0 to pH 9.0. Sporozoites were highly active over a broad range from pH 5.5 to pH 9.0, with an apparent optimum at pH 8.0. Merozoites had a much lower specific activity with pH optima at 7.0 and 8.5. The protease activity of sporozoites or merozoites could be inhibited completely by the addition of 50 micrograms/ml of leupeptin, TPCK, or TLCK or of 4 mM PMSF. Antipain inhibited proteases of sporozoites but not of merozoites. Pepstatin had little effect on either sporozoites or merozoites. The results suggest that parasite proteases of Eimeria may be necessary for invasion of host cells.     

HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA)

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.