Transcription from the Pst-Mlu fragment of LAT protects HeLa cells from anti-Fas-induced apoptosis and SY5Y cells from apoptosis induced by camptothecin.
Published data suggest that LAT is an antiapoptotic factor that may be essential for maintaining healthy neurons during latency to ultimately allow efficient reactivation under conditions of stress (19
). These data examined the effect of LAT from the McKrae and KOS viral strains on programmed cell death in tissue culture cells as well as the effect of LAT from the McKrae strain in the rabbit model.
In similarly designed experiments in which we cotransfected tissue culture cells with the plasmid pcDNA3.Pst-Mlu
expressing the Pst-Mlu
fragment of LAT (see Fig. ) together with a GFP-expressing plasmid to mark transfected cells, we observed similar results. The stable 2.0-kb LAT intron as well as exon 1 and a 5′ fragment of the exon 2 region of the LAT are expressed from this pcDNA3.Pst-Mlu
). Plasmid DNA (pcDNA3) alone was used as a negative control in these experiments, while the construct expressing the baculovirus antiapoptotic protein, p35, was used as a positive control. At 48 h posttransfection, HeLa cells were treated with the anti-Fas antibody and SY5Y cells were treated with the chemotherapeutic drug camptothecin to induce apoptosis in these cells. At several time points posttreatment, GFP-positive cells that remained adherent on the plates were counted, and the data were expressed as the percentage of GFP-positive cells that survived treatment.
In HeLa cells (Fig. ), at 24 h posttreatment with anti-Fas antibody, close to 100% of GFP-positive cells were protected from apoptosis by the p35 protein. Control (anti-Fas antibody treated) cells at this time point are mostly apoptotic, whereas prior to this time, no significant numbers of apoptotic cells are detected using the DeadEnd assay as described in the Materials and Methods (data not shown). Cells transfected with pcDNA3 showed only 10% protection from anti-Fas-induced apoptosis at 24 h, indicating that vector DNA alone is unable to prevent apoptotic cell death. However, close to 70% of cells expressing LAT from the pcDNA3 plasmid were viable. Furthermore, greater than 50% of these cells remained protected up to at least 48 h posttreatment.
FIG. 2. In vitro inhibition of apoptosis by LAT in HeLa cells (A) and in neuron-like SY5Y cells (B). Cells were transfected with 1 μg of pEGFP-C1 (GFP-expressing construct) and 3 μg of pcDNA3 vector, pcDNA3.Pst-Mlu, or pCIp35 expressing the baculovirus (more ...)
The human neuroblastoma cell line SY5Y was insensitive to apoptosis induced by anti-Fas antibody but sensitive to camptothecin. By 12 h posttreatment, control SY5Y cells treated with camptothecin showed significant apoptosis (data not shown). At this time, close to 100% of cells were protected from apoptosis in the presence of the LAT-expressing plasmid, whereas cells treated with the vector alone showed approximately 20% survival of GFP-expressing cells (Fig. ). By 24 h after treatment, the protection elicited by LAT decreased to approximately 30% but was still greater than the negative control (5%). At this time point, in both cell lines, about 90% of p35-transfected cells survived camptothecin-induced apoptosis.
These results indicate that the LAT-expressing plasmid protects cells against apoptosis induced by exogenous stimuli in both HeLa and SY5Y cells. However, the effect is not as protective as the p35 positive control.
Pst-Mlu LAT fragment blocks caspase 8-induced apoptosis in tissue culture.
To quantitatively determine whether LAT directly protects cells from apoptosis induced by caspase 8 in vitro, we cotransfected HeLa cells with a plasmid expressing an active form of caspase 8 together with the protecting plasmid and the GFP-expressing vector. Caspase 8 is a protease which is normally activated in response to extracellular stimuli by tumor necrosis factor (TNF)-like ligands to cause the formation of apoptotic cell bodies. Therefore, in these experiments, the cells expressing GFP will also be undergoing apoptosis induced by caspase 8. Thus, the protective effect of the cotransfected plasmid of interest on cells receiving apoptotic stimuli can be monitored by scoring survival of GFP-positive cells.
Figure shows pictures of GFP-expressing cells at 24 h posttransfection with pcDNA3, p35, or pcDNA3.Pst-Mlu, with or without the caspase 8-expressing construct. With vector alone (without LAT insert), in the presence of caspase 8, most of the GFP-positive cells were rounded and showed signs characteristic of apoptosis. In fact, by this time, several GFP-positive cells had rounded and lifted from the dishes. However, as expected when p35 was expressed from a cotransfected plasmid, almost all of the cells remained healthy and protected from apoptosis. By 24 h posttreatment, cells expressing LAT were also undergoing apoptosis, but not to the same extent as the negative control (as indicated in Fig. ).
FIG. 3. Inhibition of caspase 8-induced apoptosis by LAT in vitro. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3.Pst-Mlu, or pICp35 together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). The pC8 plasmid (more ...)
When these results were quantitated by counting GFP-positive cells on transfected tissue culture plates at different times posttreatment (see Materials and Methods), the results indicated that LAT expression allows a level of protection that is intermediate between the negative (vector plasmid only) control and the positive p35 control at all time points (Fig. ). Using this assay, LAT was found to be a less effective inhibitor of apoptosis than indicated in the previous experiment using anti-Fas antibody or camptothecin as the apoptotic stimulus (Fig. ) and that observed previously using other systems (36
). Therefore, it seems apparent that this decreased level of protection is the result of the different methods used to induce apoptosis.
To further verify our results, FACS analysis of GFP-positive cells was carried out in our cotransfection experiments in the presence of propidium iodide (Fig. ). To determine the percentage of cells undergoing apoptosis by caspase 8 cotransfection, the sub-G1 peak was analyzed. The data clearly indicate that cells transfected with pcDNA3.Pst-Mlu show a level of apoptosis that is between that of the negative and positive controls. The results are quantitated in Fig. . These data show that expression from the vector alone did not protect cells from apoptosis, whereas approximately 40% of cells expressing LAT were protected from apoptosis induced by caspase 8 at either 24 or 48 h. Again, as demonstrated earlier, the antiapoptotic protein p35 prevented the induction of apoptosis in these cells.
FIG. 4. FACS analysis to confirm the ability of LAT to protect cells from caspase 8-induced apoptosis. HeLa cells were transfected with 6 μg of pcDNA3, pcDNA3.Pst-Mlu, or pICp35 together with 2 μg of pCG239.GFP (membrane-bound GFP) and 1 μg (more ...) Mapping of LAT elements required for protection from apoptosis.
As mentioned previously, pcDNA3.Pst-Mlu
expresses the 2.0-kb LAT intron in addition to exon 1 and a small portion of exon 2 of the LAT. Therefore, it is possible that either the exon 1 region, the 2.0-kb intron, or the small part of exon 2 plays a role in protection from apoptosis. To determine which region of LAT contains the antiapoptotic function, mutants in the pcDNA3.Pst-Mlu
background (Fig. ) were used in an experiment similar to that in Fig. . These deletion constructs have been described previously (23
). Table summarizes the features of the exon 1, 2.0-kb LAT intron, and exon 2 regions expressed by each of the constructs (as indicated by Northern blot analysis in Fig. ).
FIG. 5. Inhibition of apoptosis by LAT mutants expressed from the pcDNA3 vector. (A) Northern blot analysis measuring the expression of LAT mutants. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3.Pst-Mlu, or the LAT mutant plasmids pΔXcm, (more ...)
The pΔXcm1 mutant contains a deletion of the 3′ half of the 2.0-kb LAT. When transfected into cells, this mutant expresses a truncated 2.0-kb LAT intron and a wild-type exon. The pΔBstE (0.9 kb) mutant contains a deletion of a portion of exon 1 as well as part of the 5′ end of the 2.0-kb LAT intron and is unable to express either the 2.0-kb LAT intron or the exon. The pΔSty vector contains a deletion of 371 of the 660 nucleotides of the LAT exon 1 region. This mutant expresses a complete 2.0-kb LAT intron but only a truncated exon. Additional mutants used in this study were the pCons and pΔCMV(Pst-Mlu
) mutants. The pCons mutant has a consensus splice branch point sequence inserted and thus produces an unstable 2.0-kb LAT intron, while the pΔCMV(Pst-Mlu
) mutant contains a deletion of the CMV promoter, thus producing no transcript (unless transcription from the LAP2 promoter is seen) (16
) and will allow determination of whether the DNA sequence itself has antiapoptotic activity.
The ability of the mutant LAT plasmids to protect cells from apoptosis induced by caspase 8 was tested and is quantitated in Fig. . The pΔCMV(Pst-Mlu
) plasmid elicited some protection at 24 h posttreatment, perhaps due to some activity in the LAP2 promoter (Fig. ) (16
). However, by 48 h, the percentage of cells that survived was close to background levels, indicating that expression of the LAT region is necessary for protection against apoptosis. The pΔXcm mutant was less effective than the wild-type 2.0-kb LAT at preventing cells from undergoing apoptosis at 24 h posttransfection. However, at later times posttreatment, this plasmid was as effective as the wild-type sequences at blocking cell death by apoptosis, indicating that the 3′ end of the 2.0-kb intron is not necessary for a protective phenotype. Nevertheless, this truncation was able to slightly modify the kinetics of the effect.
On the other hand, the mutant which did not express a complete exon 1 (pΔSty) was less protective than the wild-type LAT construct at all time points. Similarly, pΔBstE, which contains a deletion covering the 3′-terminal sequence of exon 1 and the 5′-proximal sequence of the 2.0-kb LAT intron, also shows reduced protection against the effects of caspase 8 expression. These results suggest that the exon 1 region and 5′ sequences of the 2.0-kb LAT intron are important in defending the virus against cellular apoptosis. The deletion in pΔBstE also removes the 2.0-kb LAT splice donor site and results in lack of expression of the 2.0-kb LAT intron. Therefore, it is possible that the 2.0-kb LAT intron also contributes to the antiapoptotic effect mediated by the LAT region.
In this regard it is interesting that, at the earlier time point (24 h), the plasmid expressing an unstable LAT but retaining wild-type exon and 5′ 2.0-kb LAT sequences (pCons) was less potent at preventing apoptosis than the pcDNA3.Pst-Mlu plasmid. However, at the later time points (48 and 72 h), this effect was minimal. Interestingly, in cells cotransfected with both the pΔSty mutant and the pCons mutant, the ability to protect cells from caspase 8-induced apoptosis was additive. In fact, protection was close to wild-type levels. Taken together, these results show that expression from the exon 1 region of LAT is important in maintaining the viability of cells under conditions of apoptosis. In addition, the 2.0-kb LAT intron, in particular the 5′-terminal sequences, also appears to mediate an antiapoptotic effect, although the effect is less than that mediated by exon 1.
These results (summarized in Table ), using plasmids with deletions or insertions in the LAT exon 1 or intron region, support the hypothesis that transcription of the LAT exon and the LAT 2.0-kb intron facilitate protection from apoptosis in transfected tissue culture cells. Furthermore, they show that the effects of the exon and the intron are additive and function in trans. To determine whether this effect is seen in the presence of other viral genes during the lytic cycle of virus infection, experiments were performed using virus-infected tissue culture cells.
17N/H deletion virus is less effective at preventing apoptosis induced by anti-Fas than either the wild-type or ΔSty deletion viruses in productively infected HeLa cells.
HSV-1 contains several proteins that have been shown to inhibit apoptosis during the lytic cycle of infection (20
). Therefore, to further determine the contribution of LAT as an antiapoptotic factor during the various stages of virus infection, two LAT deletion viruses, 17N/H and ΔSty, were tested for their effectiveness at preventing programmed cell death during infection of tissue culture cells and primary infection of the mouse peripheral nervous system. The ability of these mutant viruses to express exon 1, 2.0-kb LAT, and exon 2 is described in Table . The 17N/H virus (see Fig. ) does not express exon 1 or the 2.0-kb LAT intron due to an extensive deletion of the LAT promoter exon 1 and the 5′ half of the 2.0-kb LAT, but retains the LAT exon 2, whereas the ΔSty virus expresses the stable LAT intron but is deleted of most of exon 1.
In Figure , HeLa cells were infected with wild-type HSV 17, 17N/H virus, or ΔSty virus or mock infected. At 16 h postinfection, cells were either mock treated or treated with anti-Fas antibody for 6 h to induce apoptosis. After cell lysis, DNA was isolated and run on agarose gels. In mock-infected cells, in the presence of anti-Fas, there was a large amount of fragmented DNA, and in HSV 17-infected cells virus was clearly protected against apoptosis induced by anti-Fas, as expected. Furthermore, HSV 17 did not induce apoptosis in untreated cells (Fig. ).
FIG. 6. DNA fragmentation in HeLa cells productively infected with LAT deletion viruses. Mock-infected cells or cells infected with strain 17, 17N/H, or ΔSty virus (10 PFU/cell) were lysed in buffer containing 0.5% Triton X-100 and incubated for (more ...)
The 17N/H virus was less effective at protecting cells from anti-Fas-mediated apoptosis than HSV 17, its parental virus, indicating that the LAT region affected by the deletion in this virus elicits some protection against apoptosis during acute infection in tissue culture. However, in contrast to the transfection data, the ΔSty virus was as effective as strain 17 virus at preventing the onset of apoptosis. These results indicate that in the context of the lytic virus infection in tissue culture cells, the exon of the LAT is not necessary for protection against apoptosis. However, the 17N/H mutant phenotype indicates that during tissue culture infections, some region of the LAT locus, perhaps the 2.0-kb LAT or the LAT promoter region, does contribute to the inhibition of cellular apoptosis. Interestingly other transcripts have been mapped in the N/H deletion region, 1.8 kb and 1.1 kb (56
) (see Fig. ) that may play a role in the antiapoptotic effect.
LAT region of HSV-1 contributes to the viral antiapoptotic function in the acutely infected mouse peripheral nervous system.
To determine the effects of the LAT deletion viruses on apoptosis in vivo, female BALB/c mice were infected with the strain 17, 17N/H, and ΔSty viruses by corneal scarification. At 3 and 6 days postinfection, mice were sacrificed, and the trigeminal ganglia of each mouse were sectioned and processed for the presence of virus by immunohistochemistry and for apoptotic cells by the DeadEnd colorimetric assay (Promega). Although the amount of viral staining in 17N/H was low compared to that of 17 and 17ΔSty (Fig. ), the results indicate that each of the viruses replicate in the trigeminal ganglia of acutely infected mice.
FIG. 7. Detection of apoptosis in mice trigeminal ganglia. BALB/c mice were ocularly infected in both eyes with HSV 17, 17N/H, or ΔSty viruses at 5 × 104 PFU/eye. At days 3 (A) and 6 (B) postinfection, five mice were sacrificed per group, and (more ...)
Interestingly, in 17- and 17ΔSty-infected sections, we saw significant staining for HSV antigens with few apoptosis-positive staining cells associated with these regions (Fig. ). However, in 17N/H sections, we observed cells showing dual staining for HSV antigens and apoptosis (Fig. , and shown more clearly in the inset of panel Biv). Therefore, consistent with our tissue culture infection data (Fig. ), these results indicate that during the acute infection in vivo, the exon region of LAT does not exert an antiapoptotic effect. On the other hand, the N/H region of LAT appears to play a role in protecting cells from apoptosis, both in productively infected tissue culture cells and in the trigeminal ganglia of acutely infected mice.