Department of Veterinary and Biomedical Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68503, USA
Correspondence
Clinton Jones
cjones{at}unlnotes.unl.edu
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ABSTRACT |
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MAIN TEXT |
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Our previous studies demonstrated that bICP0 induced cell death in transient transfection assays (Inman et al., 2001). However, these studies did not test how bICP0 killed these cells. There are at least three forms of programmed cell death necrosis, apoptosis and paraptosis (Sperandio et al., 2000
). Caspase 3 is induced when apoptosis occurs but not when necrosis or paraptosis occurs. Two major apoptotic pathways exist in mammals the death receptor-mediated pathway and the mitochondrial pathway (Kruegger et al., 2001
; Schmitz et al., 2000
; Wang, 2001
). Regardless of which pathway is activated, caspase 3 is cleaved and activated. Consequently, testing whether bICP0 induced caspase 3 cleavage would distinguish whether the toxic effects of bICP0 were caused by apoptosis, necrosis or paraptosis. For the studies presented below, three plasmids that expressed bICP0 or mutant forms of the protein (bICP0,
bICP0 or 13G/51A; Fig. 1b
) were utilized. The respective bICP0 plasmids expressed Flag-tagged proteins in transfected mouse neuroblastoma (neuro-2A) cells that migrated as expected (Fig. 1c
). We consistently observed that slightly lower levels of wt bICP0 protein were expressed in neuro-2A cells compared with the
bICP0 and 13G/51A protein levels in transfected neuro-2A cells. Since expression of the respective bICP0 proteins was regulated by the human cytomegalovirus (CMV) promoter, subtle differences in protein expression were not related to differences in the promoters used in the respective plasmid constructs. Neuro-2A cells were used for these studies because they are derived from the peripheral nervous system (Olmsted et al., 1970
) and thus may be a good model to examine the cytotoxicity of bICP0 in neurons.
To test whether bICP0 activated caspase 3, we co-transfected neuro-2A cells with one of the bICP0 expression plasmids and the pCaspase3-Sensor plasmid (Clontech). As controls, neuro-2A cultures were co-transfected with a CMV plasmid expressing the pro-apoptotic protein Bax (Upstate Biotechnology) or a blank CMV expression vector (pcDNA3.1) and the pCaspase3-Sensor plasmid. Plasmid pCaspase3-Sensor encodes the enhanced yellow-green variant of the green fluorescent protein (GFP) and has three copies of the SV40 large T antigen nuclear localization signal at the 3' end of the GFP gene. At the 5' terminus of the GFP gene, a sequence encoding the nuclear export signal (NES) of the mitogen-activated protein kinase kinase is located. The NES is separated from GFP coding sequences by a 36 bp cassette containing the region of poly(ADP-ribose) polymerase cleaved by caspase 3. As expected, GFP was primarily localized to the cytoplasm of healthy cells (Fig. 2a). When neuro-2A cells were co-transfected with the CMV Bax expression plasmid and the pCaspase3-Sensor plasmid, caspase 3 was activated, the NES was cleaved and GFP had localized to the nucleus of transfected cells by 30 h post-transfection (Fig. 2b
). Bax was used as a positive control for apoptosis induction because it localizes to the mitochondria and induces cytochrome C and Smac/Diablo release, which results in caspase 3 cleavage and activation (Wang, 2001
). Neuro-2A cells transfected with
bICP0 (Fig. 2c
) or bICP0 (Fig. 2d
) appeared to contain more cells with GFP localized to the nucleus relative to cells transfected with the blank expression vector (Fig. 2a
).
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The results in Fig. 2 suggested that caspase 3 was activated by bICP0 and that cells transfected with the
bICP0 construct exhibited a higher frequency of caspase 3 cleavage and activation. To confirm this observation, we examined caspase 3 cleavage in transiently transfected neuro-2A cells (Fig. 3a
). Caspase 3 cleavage requires proteolytic processing at Asp-175 into a 19 kDa (pro-domain plus large fragment) and 17 kDa fragment (large fragment) (Nicholson & Thornberry, 1997
; Thornberry et al., 1997
; Wang, 2001
; Wolf & Green, 1999
). An antibody that specifically recognizes the 17 and 19 kDa fragments of cleaved caspase 3 (catalogue #9661; Cell Signalling) was used for this study. Relative to neuro-2A cultures transfected with pcDNA3.1, cultures transfected with bICP0 or
bICP0 contained higher levels of cleaved caspase 3 at 30 h post-transfection (Fig. 3a
). Cells transfected with
bICP0 or Bax contained similar levels of cleaved caspase 3. Increasing the concentration of
bICP0 plasmid did not increase the levels of cleaved caspase 3. We believe that increasing the concentration of
bICP0 led to increased cell lysis and, consequently, we were unable to observe higher levels of cleaved caspase 3 at 30 h post-transfection. The zinc RING finger mutant (13G/51A) contained lower levels of cleaved caspase 3 compared with cultures transfected with bICP0, which was consistent with the results in Fig. 2
. Neuro-2A cells transfected with 13G/51A contained higher levels of cleaved caspase 3 compared with cultures transfected with the blank expression vector. Regardless of the plasmid used for transfection, similar levels of
-actin were present in the cells at 30 h post-transfection (Fig. 3b
). bICP0 protein levels were also similar at 30 h post-transfection because the respective bICP0 constructs were cloned into a human CMV IE expression vector (Inman et al., 2001
) (for example, see Fig. 1c
).
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A low-passage-number bovine cell line, bovine turbinate (BT) cells, was also transfected with the bICP0 constructs or Bax and caspase 3 cleavage was monitored by Western blot analysis (Fig. 3d). The bICP0 constructs induced higher levels of caspase 3 cleavage in BT cells at 24 h post-transfection relative to cells transfected with the blank expression vector. At 24 h post-transfection, similar levels of
-actin and the respective bICP0 proteins were present in the samples obtained from transfected BT cells (data not shown). In contrast to the results with neuro-2A cells, the 13G/51A construct appeared to induce similar levels of cleaved caspase 3 to wt bICP0 or the
bICP0 construct, suggesting that certain cells are more sensitive to the toxic effects of bICP0. When a human cell line (293) that expresses adenovirus E1A and E1B proteins was transfected with
bICP0, an increase in cleaved caspase 3 was detected (data not shown). However, wt bICP0 did not consistently have a dramatic effect on caspase 3 cleavage. Collectively, these studies suggested that cell type-specific factors regulate the ability of bICP0 to induce caspase 3 cleavage and cell death.
Our results suggested that bICP0 was more efficient at activating caspase 3 in neuro-2A and 293 cells because
bICP0 lacked certain functions that wt bICP0 possessed. A previous study demonstrated that
bICP0 was predominantly localized to the cytoplasm of transfected neuro-2A cells, whereas bICP0 was primarily localized to the nucleus (Inman et al., 2001
), suggesting that interactions between cytoplasmic factors and bICP0 stimulated toxicity and apoptosis. The plasmid
bICP0 does not activate an HSV-1 thymidine kinase promoter (Inman et al., 2001
; Zhang & Jones, 2001
), which implies that the ability of bICP0 to activate transcription plays a role in cell survival. Finally, these studies indicated that the ability of bICP0 to activate caspase 3 cleavage occurred by an indirect mechanism, since caspase 3 cleavage was slow relative to Bax. Regardless of the mechanism by which bICP0 stimulates caspase 3 cleavage, these studies strongly suggest that the ability of bICP0 to promote caspase 3 cleavage plays an important role with respect to the toxic effects of bICP0.
During BHV-1 productive infection, caspase 3 is activated (Lovato et al., 2003), but induction of apoptosis is a relatively late event (Devireddy & Jones, 1999
), which correlates well with the finding that bICP0 activates caspase 3 cleavage more slowly than Bax. Alphaherpesviruses encode several anti-apoptotic proteins during productive infection (Aubert & Blaho, 2001
; Jones, 1998
, 2003
), suggesting that the ability of bICP0 to induce caspase 3 cleavage is delayed during productive infection. In general, it is believed that virally induced apoptosis and/or caspase activation is detrimental for virus replication and spread. However, there are examples of caspase activation promoting virus replication. For example, caspase cleavage of a non-structural protein stimulates replication of Aleutian mink disease parvovirus in cultured cells (Best et al., 2003
). Caspase 3 can also enhance HSV-1 reactivation from latency when latently infected neurons are explanted (Hunsperger & Wilcox, 2003
). Since bICP0 promotes reactivation from latency (Jones, 2003
), the ability of bICP0 to activate caspase 3 may stimulate reactivation from latency and/or productive infection.
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ACKNOWLEDGEMENTS |
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Received 16 June 2004;
accepted 16 August 2004.