| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Correspondence
Lisa O. Roberts
l.roberts{at}surrey.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Adams, 2003; Jiang & Wang, 2004). Apoptosis is characterized by a series of well-defined morphological and biochemical changes, including cell shrinkage, nuclear chromatin condensation and proteolysis of key cellular proteins by members of a highly conserved family of cysteine proteases called caspases. Activation of caspases occurs through proteolysis of the interdomain linker with the removal of the pro-domain and subsequent cleavage to yield the large and small fragments that reassociate to form the catalytically active enzyme (reviewed by Boatright & Salvesen, 2003). There are two well-defined yet cross-talking mechanisms that can induce the activation of distinct initiator caspases: the death receptor (extrinsic) and the mitochondrial (intrinsic) pathways. Several apoptogenic stimuli such as UV irradiation and drugs promote the release of factors such as cytochrome c from mitochondria (Kroemer & Reed, 2000; Green & Kroemer, 2004; Jiang & Wang, 2004). Cytochrome c interacts with the apoptotic protease-activating factor 1 (Apaf-1), which forms an essential part of the apoptosome (Jiang & Wang, 2004). Recruitment of pro-caspase-9 into the apoptosome leads to activation of caspase-9, which mediates the activation of downstream effector caspases such as caspase-3 and -7.
Members of the family Caliciviridae are responsible for a number of diseases of man and animals (reviewed by Clarke & Lambden, 2000). Feline calicivirus (FCV), a member of the genus Vesivirus, causes upper respiratory tract disease in cats. The genome is a single strand of positive-sense RNA of about 7·5 kb (Carter et al., 1992); it is polyadenylated and has a 15 kDa protein called VPg covalently linked to the 5' end (Herbert et al., 1997). The genome is organized into three open reading frames (ORFs): ORF1 encodes the non-structural proteins, ORF2 encodes the capsid protein and ORF3 encodes a small basic protein that is a minor component of virions (Sosnovtsev & Green, 2000). ORF2 and ORF3 are expressed from a subgenomic mRNA (Herbert et al., 1996).
It has been shown previously that FCV induces apoptosis in cultured cells (Al-Molawi et al., 2003; Sosnovtsev et al., 2003). Activation of the executioner caspases, caspase-3 and -7 (Al-Molawi et al., 2003), and of the initiation caspases, caspase-8 and -9 (Sosnovtsev et al., 2003) and caspase-2 (Al-Molawi et al., 2003), has been shown to occur during infection. In addition, cleavage of the 62 kDa viral capsid protein into a 40 kDa protein has been observed concomitant with the apoptotic changes (Al-Molawi et al., 2003). Although it has been shown that synthesis of virus proteins is required (Sosnovtsev et al., 2003), the molecular mechanism and especially the nature of the events leading to apoptosis are not well understood. We have sought to understand the molecular mechanism of FCV-induced apoptosis in more detail by analysing the early apoptotic events.
In order to analyse the time of activation of caspase-3, Crandell–Rees feline kidney (CRFK) cells were infected with FCV F9 at an m.o.i. of 100 p.f.u. per cell, or mock infected as described previously (Al-Molawi et al., 2003). Cells were harvested at various times following infection and caspase-3 processing was measured by flow cytometric detection of the neo-epitope generated as a result of the cleavage of pro-caspase-3 to the p17/p20 fragments by immunostaining with a phycoerythrin-conjugated anti-active caspase-3 antibody as recommended by the manufacturer (BD Pharmingen) (Macanas-Pirard et al., 2005). In parallel, cells were analysed for signs of apoptosis, as assessed by phosphatidylserine (PS) exposure on their surface. CRFK cells were infected as described above, trypsinized, centrifuged and incubated with Annexin V–FITC as recommended by the supplier (Oncogene Research Products) and propidium iodide (PI, 0·6 µg ml–1; Calbiochem/Oncogene). A minimum of 10 000 events was acquired in list mode using a Beckman Coulter Epics XL, while gating the forward and side scatters to exclude cell debris, and analysed in FL1 (Annexin V–FITC) and FL3 (PI) channels. Induction of apoptosis was detected at 8 h post-infection (p.i.) (Fig. 1a) and correlated with the appearance of activated caspase-3 (Fig. 1b). Both PS exposure and caspase-3 activation were sensitive to the pan-caspase inhibitor Z-VAD-fmk (100 µM; Bachem).
|
m). The loss of m is an early event in cells undergoing apoptosis (Decaudin et al., 1997; Bossy-Wetzel et al., 1998) and has been observed in other virus infections (Tropea et al., 1995; Eleouet et al., 1998; Summerfield et al., 1998; Jacotot et al., 2000). The mechanism is not well understood, but it is a clear indication of the involvement of mitochondria in apoptosis. m was measured by flow cytometry (data not shown) and confocal laser microscopy. For the latter, CRFK cells were grown on coverslips and infected as before. At each time point p.i., the medium was removed and replaced with Dulbecco's PBS supplemented with D-glucose. The cells were incubated with tetramethylrhodamine ethyl ester (TMRE; 100 nM) in the dark for 40 min at 37 °C and examined by confocal laser microscopy using an LSM 510 Meta (Zeiss). The correct subcellular localization of TMRE was confirmed by co-localization with MitoTracker Green FM (50 nM; both reagents from Molecular Probes). This dye selectively stains mitochondria and becomes fluorescent once it accumulates in the mitochondrial lipid environment, but, unlike TMRE, the emission does not depend on m (Fig. 2). The dependence of TMRE mitochondrial staining on m was confirmed further by showing that CRFK cells pre-treated with 10 µM of the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone failed to sequester TMRE, but showed unaltered MitoTracker Green staining (data not shown). In mock-infected cells, the presence of a high m was evidenced by an intense and punctate staining with TMRE (Fig. 2, top row). At 6 h after infection with FCV (Fig. 2, bottom row), a near complete loss of TMRE staining was recorded, demonstrating a substantial decrease in m.
|
m that probably involves the rapid opening of the mitochondrial permeability transition pore (Green & Kroemer, 2004; Jiang & Wang, 2004; Sharpe et al., 2004). We therefore analysed the location of Bax and cytochrome c during FCV infection of CRFK cells. Cells were infected as above and at the indicated time points were washed, resuspended in intracellular medium buffer (20 mM NaCl, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 23 mM HEPES, pH 7·1) supplemented with 1 µM cyclosporin A (to prevent mitochondrial permeability transition pore opening during sample processing) and permeabilized on ice for 10 min by the addition of 600 µg digitonin ml–1 (Macanas-Pirard et al., 2005). Here, digitonin was used instead of saponin (Macanas-Pirard et al., 2005) as it provided a superior concentration range of plasma membrane permeabilization and damage to the outer mitochondrial membrane (G. E. N. Kass, unpublished observations). Cells were centrifuged (2000 g for 5 min) to separate cytosolic proteins from membrane (mitochondria)-associated proteins. Equal amounts of protein from each fraction were assayed by SDS-PAGE and immunoblotting (Macanas-Pirard et al., 2005). Proteins were detected using antibodies raised against cytochrome c (clone 7H8.2C12, diluted 1 : 1000; BD Pharmingen), Bax (N-20, diluted 1 : 500; Santa Cruz Biotechnology), cytochrome c oxidase (COX subunit IV, clone 20E8-C12, diluted 1 : 1000; Molecular Probes) or Hsc70 (diluted 1 : 1000; Stressgen Biotechnologies). At 4 h p.i., the localization of cytochrome c was still mitochondrial [(Fig. 3a, P (particulate)]. The appearance of cytochrome c in the cytosolic fraction (Fig. 3a, S) was clearly detected by 6 h and was almost complete at 8 h, therefore preceding the processing of caspase-3 and PS exposure. The release of cytochrome c also correlated with the translocation of the pro-apoptotic Bcl-2 protein family member, Bax, from the cytosol to the mitochondria at 6 h p.i. (Fig. 3b). The detection of COX was used as a control for the cytosolic fraction to demonstrate the lack of contamination by mitochondria, whereas Hsc70 was used as a loading control for the cytosolic fraction. At 8 h p.i., we detected a decrease in the amount of Hsc70 in the cytosol (Fig. 3b); however, this was probably due to the general permeabilization of the plasma membrane late in infection.
|
). Recruitment of pro-caspase-9 into the apoptosome leads to activation of caspase-9, which mediates the activation of downstream caspases such as caspase-3. Therefore, we analysed the timing of pro-caspase-9 processing during FCV infection. Cells were infected as described above and cell lysates were subjected to SDS-PAGE and immunoblotting with anti-caspase-9 antisera (a gift from Professor Yuri Lazebnik, Cold Spring Harbor Laboratory, USA; Rodriguez & Lazebnik, 1999). Activation of caspase-9, as measured by the disappearance of the 47 kDa pro-form, was clearly seen by 6 p.i. (Fig. 3c
), which coincided with the release of cytochrome c, but preceded caspase-3 activation (Fig. 1). Activation of caspase-9 was also measured using an enzymic assay (Caspase-Glo 9; Promega). Briefly, cells were collected 8 h after infection with FCV and incubated with Caspase-Glo 9 reagent as recommended by the manufacturer. Relative light units (RLU) were read using a luminometer (LumiCount; Packard Bioscience). This method demonstrated a large increase in caspase-9 activity at 8 h p.i., in line with the increase in activity seen in CRFK cells treated with staurosporine (STS, 1 µM for 8 h) (Fig. 3d).
During infection, CRFK cells present the hallmarks of apoptosis–chromatin condensation, poly(ADP-ribose) polymerase cleavage, DNA fragmentation and caspase activation. In the present study, we extended these observations and studied events upstream of caspase-3 activation to define the molecular mechanism of FCV-induced apoptosis. Consistent with previous reports, we showed that the executioner caspase, caspase-3, was activated at 8 h after infection with FCV (Al-Molawi et al., 2003; Sosnovtsev et al., 2003). This correlated with the onset of apoptosis at 8 h p.i., as demonstrated by PS exposure on the cell surface. The pan-caspase inhibitor Z-VAD-fmk prevented caspase-3 activation and inhibited apoptosis. This demonstrated that, in FCV infection, the execution of apoptosis is dependent on caspases. In order to understand the molecular events leading to caspase-3 activation in FCV infection, we analysed early apoptotic events preceding caspase-3 activation. We saw a loss in m from 6 h p.i. and this coincided with the translocation of Bax to, and the release of cytochrome c from, mitochondria. The release of cytochrome c was paralleled by caspase-9 processing and only at 8 h when it was completed did it correspond to the time of activation of caspase-3. It has been reported that cytochrome c can be released from mitochondria during the extrinsic pathway as a result of the cleavage of Bid by caspase-8 (McDonnell et al., 2003). However, this is unlikely to explain events in our system, since the caspase-8 inhibitor acetyl-Ile-Glu-Thr-Asp-aldehyde (100 µM) failed to inhibit or delay FCV-induced apoptosis (data not shown). These results strongly suggest that the mitochondrial pathway of apoptosis is triggered in FCV infection.
The events upstream of Bax translocation to mitochondria in FCV-infected cells are currently under investigation. However, many other RNA viruses can trigger the mitochondrial pathway of apoptosis in infected cells. For example, poliovirus (PV) has been shown to induce apoptosis through this pathway, inducing cytochrome c release and activation of caspase-9 (Belov et al., 2003). Furthermore, expression of PV 3C protease in cells can trigger apoptosis (Barco et al., 2000). FCV protease shares features with the picornavirus superfamily 3C proteases and the potential role of this protein in FCV-triggered apoptosis is being investigated. Similarly, the 2C protein of avian encephalomyelitis virus has been shown to trigger apoptosis through the mitochondrial pathway (Liu et al., 2004). The FCV p39 protein contains the NTP-binding domain conserved in picornavirus 2C proteins and it is attractive to speculate that it may act in a similar manner to the picornavirus 2C protein. However, it is possible that more than one FCV protein may be responsible for triggering apoptosis. While it is clear that FCV-infected cells undergo apoptosis, the biological consequences of this process in terms of virus replication are not known. It has been demonstrated that inhibition of apoptosis during astrovirus infection drastically decreases the amount of virus released from cells (Guix et al., 2004), while inhibition of apoptosis in vesicular stomatitis virus infection has no effect on replication (Hobbs et al., 2003). Understanding the molecular mechanisms of apoptosis triggered in calicivirus infection may also be important in understanding the progression of disease in the animal. In addition, using FCV as a model may shed light on the mechanism of cell destruction in human calicivirus infections. It has been reported that calicivirus infections in intestinal transplant patients give rise to similar apoptotic features to those seen in allograft rejection (Kaufman et al., 2003; Morotti et al., 2004) and therefore infection can be mistaken for rejection. Thus, it may be possible to define the specific apoptotic changes triggered during infection to help distinguish between infection and rejection.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Al-Molawi, N., Beardmore, V. A., Carter, M. J., Kass, G. E. N. & Roberts, L. O. (2003). Caspase-mediated cleavage of the feline calicivirus capsid protein. J Gen Virol 84, 1237–1244.
Arends, M. J. & Wyllie, A. H. (1991). Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32, 223–254.[Medline]
Barco, A., Feduchi, E. & Carrasco, L. (2000). Poliovirus protease 3Cpro kills cells by apoptosis. Virology 266, 352–360.[CrossRef][Medline]
Belov, G. A., Romanova, L. I., Tolskaya, E. A., Kolesnikova, M. S., Lazenik, Y. A. & Agol, V. I. (2003). The major apoptotic pathway activated and suppressed by poliovirus. J Gen Virol 77, 45–56.
Boatright, K. M. & Salvesen, G. S. (2003). Mechanisms of caspase activation. Curr Opin Cell Biol 15, 725–731.[CrossRef][Medline]
Bossy-Wetzel, E., Newmeyer, D. D. & Green, D. R. (1998). Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17, 37–49.[CrossRef][Medline]
Carter, M. J., Milton, I. D., Meanger, J., Bennett, M., Gaskell, R. M. & Turner, P. C. (1992). The complete nucleotide sequence of a feline calicivirus. Virology 190, 443–448.[CrossRef][Medline]
Clarke, I. N. & Lambden, P. R. (2000). Organization and expression of calicivirus genes. J Infect Dis 181, S309–S316.[CrossRef][Medline]
Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R. & Kroemer, G. (1997). Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 57, 62–67.
Eleouet, J. F., Chilmonczyk, S., Besnardeau, L. & Laude, H. (1998). Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway. J Virol 72, 4918–4924.
Green, D. R. & Kroemer, G. (2004). The pathophysiology of mitochondrial cell death. Science 305, 626–629.
Guix, S., Bosch, A., Ribes, E., Martinez, L. D. & Pintó, R. M. (2004). Apoptosis is astrovirus-infected CaCO-2 cells. Virology 319, 249–261.[CrossRef][Medline]
Herbert, T. P., Brierley, I. & Brown, T. D. K. (1996). Detection of the ORF3 polypeptide of feline calicivirus in infected cells and evidence for its expression from a single, functionally bicistronic, subgenomic mRNA. J Gen Virol 77, 123–127.
Herbert, T. P., Brierley, I. & Brown, T. D. K. (1997). Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation. J Gen Virol 78, 1033–1040.[Abstract]
Hobbs, J. A., Hommel-Berrey, G. & Brahmi, Z. (2003). Requirement of caspase-3 for efficient apoptosis induction and caspase-7 activation but not viral replication or cell rounding in cells infected with vesicular stomatitis virus. Hum Immunol 64, 82–92.[CrossRef][Medline]
Jacotot, E., Ravagnan, L., Loeffler, M. & 15 other authors (2000). The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 191, 33–45.
Jiang, X. J. & Wang, X. D. (2004). Cytochrome c-mediated apoptosis. Annu Rev Biochem 73, 87–106.[CrossRef][Medline]
Kaufman, S. S., Chatterjee, N. K., Fuschino, M. E. & 9 other authors (2003). Calicivirus enteritis in an intestinal transplant recipient. Am J Transplant 3, 764–768.[CrossRef][Medline]
Kroemer, G. & Reed, J. C. (2000). Mitochondrial control of cell death. Nat Med 6, 513–519.[CrossRef][Medline]
Liu, J., Wei, T. & Kwang, J. (2004). Avian encephalomyelitis virus nonstructural protein 2C induces apoptosis by activating cytochrome c/caspase-9 pathway. Virology 318, 169–182.[CrossRef][Medline]
Macanas-Pirard, P., Yaacob, N. S., Lee, P. C., Holder, J. C., Hinton, R. H. & Kass, G. E. N. (2005). Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells. J Pharmacol Exp Ther 313, 780–789.
McDonnell, M. A., Wang, D., Khan, S. M., Vander Heiden, M. G. & Kelekar, A. (2003). Caspase-9 is activated in a cytochrome c-independent manner early during TNF alpha-induced apoptosis in murine cells. Cell Death Differ 10, 1005–1015.[CrossRef][Medline]
Morotti, R. A., Kaufman, S. S., Fishbein, T. M., Chatterjee, N. K., Fuschino, M. E., Morse, D. L. & Magid, M. S. (2004). Calicivirus infection in pediatric small intestine transplant recipients: pathological considerations. Hum Pathol 35, 1236–1240.[CrossRef][Medline]
Rodriguez, J. & Lazebnik, Y. (1999). Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev 13, 3179–3184.
Sharpe, J. C., Arnoult, D. & Youle, R. J. (2004). Control of mitochondrial permeability by Bcl-2 family members. Biochim Biophys Acta 1644, 107–113.[Medline]
Sosnovtsev, S. V. & Green, K. Y. (2000). Identification and genomic mapping of the ORF3 and VPg proteins in feline calicivirus virions. Virology 277, 193–203.[CrossRef][Medline]
Sosnovtsev, S. V., Prikhod'ko, E. A., Belliot, G., Cohen, J. I. & Green, K. Y. (2003). Feline calicivirus replication induces apoptosis in cultured cells. Virus Res 94, 1–10.[CrossRef][Medline]
Summerfield, A., Knotig, S. M. & McCullough, K. C. (1998). Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. J Virol 72, 1853–1861.
Tropea, F., Troiano, L., Monti, D. & 10 other authors (1995). Sendai virus and herpesvirus type-1 induce apoptosis in human peripheral blood mononuclear cells. Exp Cell Res 218, 63–70.[CrossRef][Medline]
Received 2 August 2005;
accepted 12 October 2005.
This article has been cited by other articles:
|
|
 |
|
  D. Bailey, W. J. Kaiser, M. Hollinshead, K. Moffat, Y. Chaudhry, T. Wileman, S. V. Sosnovtsev, and I. G. Goodfellow Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation J. Gen. Virol., March 1, 2010; 91(3): 739 - 749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bok, V. G. Prikhodko, K. Y. Green, and S. V. Sosnovtsev Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin J. Virol., April 15, 2009; 83(8): 3647 - 3656. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Martin-Latil, L. Mousson, A. Autret, F. Colbere-Garapin, and B. Blondel Bax Is Activated during Rotavirus-Induced Apoptosis through the Mitochondrial Pathway J. Virol., May 1, 2007; 81(9): 4457 - 4464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Ossiboff, A. Sheh, J. Shotton, P. A. Pesavento, and J. S. L. Parker Feline caliciviruses (FCVs) isolated from cats with virulent systemic disease possess in vitro phenotypes distinct from those of other FCV isolates J. Gen. Virol., February 1, 2007; 88(2): 506 - 517. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
