Department of Molecular Biosciences, University of Kansas, 7047 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, KS 66045-7534, USA
Correspondence
Sandra Quackenbush
squack{at}ku.edu
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ABSTRACT |
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Present address: Stowers Institute for Medical Research, Kansas City, MO, USA.
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Introduction |
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WDSV contains three open reading frames (ORFs) in addition to gag, pol and env, which have been designated orf a, orf b and orf c. orf a and orf b are downstream of env, in the 3' proximal region of the genome, while orf c lies between the 5' long terminal repeat and gag (Holzschu et al., 1995). The location and sequence of orf c is conserved in two other members of the genus Epsilonretrovirus, walleye hyperplasia virus types 1 and 2 (WEHV-1 and -2) (LaPierre et al., 1999
). Northern blot analysis revealed subgenomic viral transcripts, encoding orf a and orf b, in developing as well as regressing tumours, but only regressing tumours contain full-length genomic transcripts, encoding orf c, and these transcripts are very abundant (Bowser et al., 1996
; Quackenbush et al., 1997
). Likewise, orf c is encoded in the genomic transcripts of WEHV-1 and -2 (LaPierre et al., 1999
). The predicted gene product of orf c is a very basic protein, 120 aa in length, with a predicted molecular mass of 14·1 kDa. LaPierre et al. (1999)
identified a possible WW domain (WX42WX36WX41W) in the Orf C proteins of walleye retroviruses and, though the WDSV Orf C protein is divergent (WX42YX36WX33W), Orf C interaction with proteins containing proline-rich regions is possible. To date, no other protein homologues for Orf C have been identified.
The position of orf c, before the initiation of gag in viral transcripts, poses many interesting questions regarding its functional role in conjunction with structural proteins and virus particle morphogenesis and budding. To further our understanding of the function of WDSV Orf C, cell fractionation and immunofluorescence microscopy were used to determine the subcellular distribution of expressed Orf C.
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Methods |
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Cells and transfection.
Mammalian cell lines Cf2Th [canine thymus, ATCC CRL1430 (Nelson-Rees et al., 1976)], HeLa [human carcinoma, ATCC CCL 2 (Gey et al., 1952
)] and NIH 3T3 [mouse fibroblast, ATCC CRL 1658 (Andersson et al., 1979
; Copeland & Cooper, 1979
; Jainchill et al., 1969
)] were maintained at 37 °C with 5 % CO2 in DMEM supplemented with 5 % foetal bovine serum (FBS), 2 mM glutamine, 100 units penicillin ml-1 and 100 µg streptomycin ml-1. Immortalized WF-2 walleye fry fibroblasts and primary W12 walleye fibroblasts were maintained in MEM with Hanks' salts and 10 % FBS at 20 °C. WF-2 cells were derived from whole walleye fry in the laboratory of Bruce Calnek, Cornell University, NY, USA (Wolf & Mann, 1980
). W12 cells were derived from walleye dermal sarcomas in the laboratory of Paul Bowser, Cornell University, NY, USA. They do not harbour WDSV provirus (Rovnak et al., 2001
). For Western blot analysis, subconfluent monolayers in 35 mm dishes were transfected with 2 µg plasmid pKH3OrfC or control vectors using FuGENE6, according to the manufacturers' instructions (Boehringer Mannheim/Roche). Alternatively, cells grown on 5 mm spots on glass slides (Cel-Line) were transfected with 50 ng DNA.
Immunoblotting.
Regressing tumours were excised from adult animals during the spawning season and stored at -80 °C. Whole tumours were extracted twice for 30 min at 4 °C in an equal volume of buffer containing 0·5 % sodium deoxycholate, 0·1 % SDS, 1 % NP-40, 150 mM NaCl, 50 mM Tris (pH 8·0), 0·2 mM sodium orthovanadate, 0·2 mM PMSF, 2 µg leupeptin ml-1, 2 µg aprotinin ml-1 and 1 µg pepstatin ml-1. Alternatively, skin was scraped from frozen, uninfected fingerling fish for extraction. Extracts were pooled and their protein concentration determined by BCA (bicinchoninic acid) (Pierce). For Western blots of tumour or skin extract, 150 µg protein was separated on a 12 % NuPAGE gel (Invitrogen) and transferred to an Immobilon-P membrane (Millipore). The membrane was blocked in Blotto (5 % nonfat dry milk and 0·5 % Tween 20 in PBS) and incubated overnight with a 1 : 250 dilution of anti-Orf C sera. Blots were washed, incubated with a 1 : 5000 dilution of affinity-purified goat anti-rabbit IgG conjugated with horseradish peroxidase (Kirkegaard & Perry Laboratories) and developed with the substrate 3,3',5,5'-tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories). The blots were then stripped and reprobed with a 1 : 250 dilution of anti-WDSV capsid. For analysis by immunoprecipitation, aliquots of 500 µg protein at 1 µg µl-1 were precleared with protein GSepharose (Amersham Pharmacia) for 1 h at 4 °C, then incubated overnight with 2·5 µl preimmune, control rabbit sera or with rabbit sera reactive to Orf C or capsid proteins (generous gifts from Volker M. Vogt, Cornell University, NY, USA). Immune complexes were precipitated for 4 h with 25 µl protein GSepharose suspension and precipitates were washed four times in lysis buffer, heated in 20 µl sample buffer and analysed by Western blot, as above, for detection of immunoprecipitated Orf C.
Transfected cells were lysed with 500 µl buffer containing 1 % Triton X-100, 0·5 % NP-40, 150 mM NaCl, 10 mM Tris (pH 7·5), 1 mM EDTA (pH 8·0), 1 mM EGTA (pH 8·0), 0·2 mM sodium orthovanadate, 0·2 mM PMSF, 2 µg leupeptin ml-1, 2 µg aprotinin ml-1 and 1 µg pepstatin ml-1. Lysates were centrifuged at 21 000 g for 15 min and the supernatant and pellet were analysed separately. Pellets were suspended in 20 µl PBS. A 20 µg sample of lysate or 10 µl of pellet suspension was separated on a 10 % NuPAGE gel and transferred to a an Immobilon-P membrane. The membrane was blocked in Blotto and incubated with a monoclonal antibody (mAb) that recognizes the HA epitope (12CA5). Blots were washed, incubated with a 1 : 5000 dilution of affinity-purified goat anti-mouse IgG conjugated with horseradish peroxidase and developed with the substrate TMB.
Fluorescence microscopy.
Cells grown on slides and transfected with pKH3OrfC were fixed in 2 % buffered paraformaldehyde for 30 min, rinsed in PBS, permeabilized with 1 % Triton X-100 in PBS for 15 min, washed three times with PBS, rinsed in water and air dried. Fixed cells were incubated with a 1 : 1000 dilution of anti-HA mAb (HA.11, clone 16B12; Covance) for 1 h at 37 °C in a humidified chamber. Slides were then washed in PBS and incubated with a 1 : 40 dilution of goat anti-mouse IgG conjugated with FITC or rhodamine (Kirkegaard & Perry Laboratories). After washing in PBS, cells were counterstained with 300 µM 4',6'-diamidino-2-phenylindole (DAPI). For colocalization, cells were incubated with rabbit anti-HA (HA.11) and mouse mAb reactive to cytochrome c (clone 6H2.B4; Pharmingen), followed by a mixture of goat anti-rabbit IgGFITC and goat anti-mouse IgGrhodamine. For detection of apoptosis, live cells on slides were stained with 65 µg Annexin VFITC ml-1 (Santa Cruz Biotechnologies) in Annexin assay buffer [10 mM HEPES (pH 7·4), 140 mM NaCl and 25 mM CaCl2) for 15 min at room temperature. Cells were then fixed and stained as above with anti-HA antibody. Control cells were transfected with a WDSV Orf A expression vector, pKH3OrfA. Mitochondria were stained in live cells with 100 nM Mitotracker Orange (CMTMRos, Molecular Probes) in complete media at 37 °C for 1 h prior to fixing. All images were viewed on a Ziess Axiophot microscope and captured in colour with an Optronics camera and MAGNAFIRE software. Images were overlaid digitally with Adobe Photoshop, version 4.0.
Cell fractionation.
The ApoAlert Cell Fractionation kit (Clontech) was used, according to manufacturer's instructions, to separate a highly enriched mitochondrial fraction from the cytosol of cells transfected with pKH3OrfC or control vector pCMVHA. Briefly, transfected cells were homogenized and fractionated by means of differential centrifugation. The protein concentration of the cytosolic and mitochondrial fractions was determined with the Bio-Rad Protein Assay kit. Immunoblotting was performed as described above. Blots were incubated with either anti-HA (12CA5 mAb; 1 : 1000), anti-COX4 (Clontech mAb; 1 : 500) or anti-cytochrome c (Clontech rabbit antisera; 1 : 100).
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Results |
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Discussion |
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When expressed in tissue culture cells, WDSV Orf C was located in punctate, cytoplasmic structures and 26 % of Orf C-containing cells were positive for Annexin V staining, indicative of apoptosis. A large portion of Orf C-positive cells were degraded to such a degree as to make their Annexin V status unclear. The identity of the cytoplasmic structures that concentrate Orf C was determined by colocation with cytochrome c in mitochondria. The association of Orf C with mitochondria was confirmed using subcellular fractionation studies, in which Orf C was only detected in the highly enriched mitochondrial fraction. Other retroviral proteins, known to be targeted to the mitochondria, are Vpr of human immunodeficiency virus (HIV) and p13II of human T-lymphotropic virus type I (HTLV-I) (Ciminale et al., 1999; Jacotot et al., 2000
). The mechanism by which Orf C is targeted to the mitochondria is unknown. Computer analysis of the Orf C amino acid sequence using TARGETP software (Emanuelsson et al., 2000
) does not identify a mitochondrial or other targeting sequence. Orf C, like HTLV p13II, may contain a novel mitochondrial-targeting sequence (Ciminale et al., 1999
) or may bind directly to other mitochondrial proteins. Of the viral proteins that are targeted to the mitochondria, all lack the canonical amino-terminal mitochondrial localization sequence and may utilize membrane insertion pathways (Boya et al., 2001
). The tight association of Orf C with mitochondria may be mediated by proteinprotein interactions via its putative WW domain (LaPierre et al., 1999
) or Orf C may interact with the acidic lipids of the inner mitochondrial membrane due to its predicted hydrophobic and basic regions.
Immunofluorescence staining with anti-cytochrome c demonstrated that the distribution of mitochondria was severely affected in Orf C-expressing cells. Mitochondria are normally dispersed throughout the cytoplasm in a distinct pattern due to their association with microtubules and control cells in the present study exhibited this pattern of mitochondrial distribution. However, perinuclear clustering of mitochondria was observed consistently in cells expressing Orf C. Perinuclear clustering of mitochondria has also been observed in cells expressing HIV Vpr, HTLV p13II and hepatitis B virus (HBV) HBx (Ciminale et al., 1999; Jacotot et al., 2000
; Takada et al., 1999
). De Vos et al. (1998)
demonstrated that stimulation of cells with tumour necrosis factor (TNF) induced perinuclear clustering of mitochondria and subsequent apoptosis and that these effects are attributed to impairment of kinesin motor activity (De Vos et al., 2000
).
The effects of the Orf C localization were revealed further by the inability of mitochondria in these cells to accumulate the MitoTracker dye. In normal cells, as in control transfects, the intensity of fluorescence of the MitoTracker was quite intense, but in Orf C-positive cells fluorescent marking of mitochondria was lost. Orf C shares this ability with HIV Vpr, HBV HBx and HTLV p13II (Ciminale et al., 1999; Jacotot et al., 2000
; Rahmani et al., 2000
). Loss of accumulation of MitoTracker is associated with dissipation of
m, an early event in apoptosis and an indicator of the opening of a conductance channel, called the permeability transition pore (PTP). Opening of the PTP leads to matrix swelling and eventual rupture of the outer membrane, allowing release of cytochrome c, apoptosis-inducing factor and caspases. Dispersal of cytochrome c in the cytosol of Orf C-expressing cells was not clear by Western blot analysis. However, we were able to detect diffuse fluorescent staining of cytochrome c in a few cells and there was an apparent decline in cytochrome c levels in the mitochondrial fraction of Orf C transfects. Our results may be limited by the transient transfection used in these assays. Establishment of a stable cell line with inducible Orf C will allow temporal analysis of
m loss and release of cytochrome c. Studies with HTLV p13II also demonstrated alteration of
m without substantial release of cytochrome c (Ciminale et al., 1999
) and HIV Vpr disrupts
m before there is a detectable release of cytochrome c to the cytosol (Jacotot et al., 2001
). Vpr interacts directly with the adenine nucleotide translocator, a component of the PTP, to form a conductance channel that permeabilizes the inner membrane before the outer membrane becomes permeable to cytochrome c (Jacotot et al., 2001
). Investigation of the functional effects of Orf C on mitochondria revealed that Orf C expression was associated with a disruption of the mitochondrial membrane potential.
In this study we demonstrate that WDSV Orf C is targeted to the mitochondria and that this localization results in perinuclear clustering of mitochondria and probable dissipation of m. The expression of Orf C in cell culture resulted ultimately in apoptosis and suggested that Orf C plays a role as a mediator of cell death during the process of WDSV replication. It has been postulated that Orf C is synthesized from full-length, genomic RNA in conjunction with viral structural proteins and full-length transcripts have only been detected in regressing tumours (Bowser et al., 1996
; Martineau et al., 1992
; Quackenbush et al., 1997
). It has been suggested also that walleye dermal sarcoma regression is mediated by apoptosis (Lairmore et al., 2000
; LaPierre et al., 1998
; Zhang & Martineau, 1999
). High numbers of apoptotic cells are detectable in tumour sections by use of the TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling) to detect DNA fragmentation (J. Rovnak, unpublished). Activation of apoptosis at the time of, or just prior to, spawning may represent an important step in the biology of walleye dermal sarcoma, possibly by contributing to the spread of infectious virus to naive animals while limiting the induction of inflammatory and immune responses.
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ACKNOWLEDGEMENTS |
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Received 11 May 2002;
accepted 22 October 2002.