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*
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195;
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and
Evanston Northwestern Healthcare Research Institute, Evanston, IL 60201
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100 million Americans and
cause significant morbidity. The clinical significance of these
infections should not be underestimated; neonatal herpes occurs in >11
of every 100,000 live births (1), and genital HSV disease
is an important cofactor in HIV transmission (2). Host
immune defenses are critical in the control of HSV infections, and
severe disease is seen in patients with impaired cellular immunity.
Even in the setting of an intact immune system, control of HSV is
incomplete, and recurrences are common. Because there is no evidence that HSV uses antigenic variation to escape host control, it must use alternative immune escape strategies if it is to successfully reactivate and be transmitted. A major evasion mechanism used by HSV is the establishment of latency in the dorsal root ganglion. The virus can persist and avoid the host immune system, yet periodically reactivate, causing shedding and lesions. However, during the reactivation process, the virus must face the host defenses, and thus a new set of countermeasures emerges. For example, when the virus reactivates and infects dermal fibroblasts and keratinocytes, it causes down-regulation of MHC class I via inhibition of TAP by ICP47 (3, 4), thus interfering with the recognition of these cells by CD8+ CTL (5).
We recently demonstrated a second mechanism by which HSV may blunt the attack of CTL, in which HSV protects infected cells from CTL-induced apoptosis (6). Because CTL kill their targets at least in part by inducing apoptosis, an apoptosis-resistant phenotype of HSV-infected cells could constitute a strategy of immune evasion. Studies of other viruses show that several encode genes that inhibit the Fas pathway of apoptosis induction by CTL (7). CTL of the mammalian immune system have evolved another mechanism of apoptosis induction via exocytosis of lytic granules. The granule exocytosis pathway appears to be the major pathway for cytotoxicity induced by HSV-specific human CTL (8). A number of cytotoxic molecules are present in the lytic granule and are involved in the induction of target cell death (9). Granzyme (gr)3B appears to be especially important in this regard and shows the most rapid kinetics of apoptosis induction, although other granule components can induce death in the absence of grB (10, 11). Through their lytic granules, CTL bypass viral inhibitors that might successfully antagonize death receptors such as Fas. The development of grB appears to have been a successful countermeasure against Fas antagonists, and inhibitors of death receptors do not antagonize grB-induced death (12).
Several genes of HSV have been shown to be necessary for the inhibition of apoptosis in various experimental systems, including Us3, Us5, ICP22, and ICP27. The usual approach in these studies has been to use mutant viruses deleted for individual HSV genes and compare these with viruses in which the deleted gene has been restored. One limitation of this approach is that although the deleted genes can be demonstrated to be necessary for efficient viral inhibition of apoptosis, the results do not imply direct anti-apoptotic activity for the deleted gene products themselves. For example, the deleted gene might instead be required for the efficient expression of some other viral gene, which was, in fact, responsible for the anti-apoptotic effect.
In this paper we demonstrate that the expression of glycoprotein J (gJ) confers apoptosis resistance upon Us5-transfected cells, confirming that this gene product is sufficient for an anti-apoptotic phenotype. Furthermore, we demonstrate that HSV antagonizes both the Fas and grB mechanisms of apoptosis induction. Us5/gJ is sufficient for the inhibition of these pathways, although in its absence other viral gene products can at least partially compensate. These data indicate that other, as yet undefined, genes are involved in the inhibition of CTL-induced apoptosis and emphasize the importance of this immune evasion strategy to the virus. Methods to circumvent this immune evasion strategy might be of significant clinical benefit to HSV-infected individuals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat and Vero cells were obtained from American Type Culture Collection (Manassas, VA). Cultured cell lines were screened regularly for mycoplasma by the Biologics Production Facility at the Fred Hutchinson Cancer Research Center (Seattle, WA).
Viruses
Laboratory virus strains E115 (HSV-1) and 333 (HSV-2), and clinical isolates F28700 (HSV-1) and T75419 and F43031 (HSV-2) were grown in Vero cells, and titers were determined using standard plaque assays. The mutant HSV-1 strains RAS116 and RAS137 contain deletions and rescues of Us5, respectively (13). The identities of the deletion and rescue virus were confirmed by PCR using Us5-specific primers.
Morphological assay using Hoechst 33342 and ethidium bromide
Morphological changes in apoptosis were observed using ethidium bromide/Hoechst 33342 staining and fluorescent microscopy (model 9901, Zeiss, New York, NY). Jurkat cells were split into log phase and incubated at 37°C for 12 h. Cells were then infected with virus at 10 PFU/cell or mock-infected and incubated at 37°C for 5 h. Infected cells were induced into apoptosis by either UV light (30-W UV bulb at 20 cm for 30 s) or anti-Fas Ab CH-11 (Tanvera, Madison, WI) at 100 ng/ml and incubated at 37°C for 4 h. A minimum of 200 cells were counted from each sample by a blinded observer and were categorized into three groups by morphological characteristics. Live cells showed no characteristics of apoptosis. Apoptotic cells showed membrane shrinkage, membrane blebbing, and/or nuclear fragmentation. Necrotic cells did not show apoptotic morphology, but allowed entry of ethidium bromide. Results were analyzed with the two-tailed Student t test using the statistical package in Microsoft Excel (Redmond, WA).
Fluorometric assay for caspase activation
Jurkat cells (1.0 x 106) were infected with HSV at 5 or 10 PFU/cell or mock-infected and incubated at 37°C for 5 h. Cells were then induced into apoptosis with UV irradiation or anti-Fas Ab and incubated for 4 h at 37°C. The cells were harvested, and lysates were evaluated for caspase activity as described previously (13), using fluorogenic substrates specific for caspase 3, 6, 8, or 9 (Calbiochem, San Diego, CA). Fluorescence was measured at 0, 30, and 60 min after addition of substrate on a PerSeptive Biosystems (Framingham, MA) CytoFluor II fluorescence plate reader. To allow comparison between separate experiments that may have different maximal relative fluorescence unit measurements, the relative fluorescence unit value for control (uninfected) cells induced into apoptosis measured at 60 min was converted to a relative activity value of 100, and other values within the same experiment were normalized to obtain a relative (percent) activity.
Regulated expression of Us5
The Us5 open reading frame from HSV-1 (17+) was amplified by PCR and cloned into the pTRE expression plasmid (CLONTECH Laboratories, Palo Alto, CA) to construct pTRE-Us5. Tet-Off Jurkat cells (CLONTECH Laboratories) were transfected with pTRE-Us5, and after selection in hygromycin and cloning, Us5 expression was either allowed or repressed with 0.5 µg/ml doxycycline. Expression of Us5 and its regulation by doxycycline were confirmed by RT-PCR (Cells-to-DNA, Ambion, Austin, TX).
Flow cytometry for mitochondrial transmembrane potential and apoptosis
Us5-tranfected cells with protein expression induced or uninduced were induced into apoptosis as described above or were mock-treated. For evaluation of mitochondrial transmembrane potential, after 5 h incubation, cells were stained for 30 min with 2.0 µg/ml 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidozolylcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR). JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (525 nm) to red (590 nm). At normal mitochondrial potential, red fluorescent J-aggregates predominate. However, as mitochondrial potential drops, the aggregates separate, and the green fluorescent monomeric form predominates. JC-1-stained cells were analyzed for relative red (FL2) to green (FL1) fluorescence, using a BD Biosciences (San Jose, CA) FACSCalibur flow cytometer (488-nm argon laser excitation source).
For analysis of permeability changes in apoptosis, cells were treated as described above and then stained with propidium iodide (PI)/YO-PRO-1 (Vybrant Apoptosis Assay kit 4; Molecular Probes) according to the manufacturer’s directions. Stained cells were analyzed for red (FL2) and green (FL1) fluorescence, using a BD Biosciences FACSCalibur flow cytometer (488-nm argon laser excitation source). Apoptotic cells show increased permeability to YO-PRO-1 (FL1 high) while remaining impermeable to PI (FL2 low).
For analysis of caspase activation in intact cells, cells were treated as described above and incubated with D2R (14) for 20 min at 37°C in the dark using the CaspSCREEN flow cytometric apoptosis detection kit (Chemicon, Temecula, CA) according to the manufacturer’s directions. Green (FL1) fluorescence was evaluated using a BD Biosciences FACSCalibur flow cytometer (488-nm argon laser excitation source).
grB activation of caspases in mitochondria-free lysates
Log phase cells were pelleted at 1200 rpm for 5 min at 4 C, washed in ice-cold PBS, and resuspended in ice-cold buffer (50 mM KCl, 50 mM HEPES (pH 7.4), 2 mM MgCl2, 1 mM DTT, 1 mM EGTA, and 1 mM PMSF) at 1 x 106 cells/ml. Cells were lysed by one cycle of freeze/thawing (-80°C/room temperature) and centrifuged 15 min (1,000 x g) at 4°C to pellet nuclei and debris. Supernatants were then centrifuged for 1 h at 100,000 x g at 4°C and either aliquoted into 96-well plates (96.5 µl/well) for immediate use or frozen in liquid nitrogen. For induction and measurement of caspase activity, 0.375 µM purified grB (Alexis) and 10 µg/ml DEVD-AMC (Alexis) were added to each well. Fluorescence was measured at time zero and after 30- and 60-min incubation at 37°C, using a CytoFluor II fluorescence plate reader.
grB introduction into intact cells using perforin
The sublytic dose of perforin (10–20% permeabilization to PI at 2 h) was determined by flow cytometry on the day of the assay. Log-phase cells were washed twice to remove serum and were resuspended in 500 µl RPMI/1% BSA at 1 x 106 cells/ml. High Ca2+ HEPES (250 µl; 10 µg/ml BSA in 20 mM HEPES, 5 mM CaCl2, and 150 mM NaCl, pH 7.4) buffer was added, followed by purified grB (1 µg/ml). The sublytic dose of perforin was added in 250 µl Ca2+-free buffer (10 µg/ml BSA in 20 mM HEPES and 150 mM NaCl, pH 7.4). Apoptosis of cells was determined 2 h later using PI/YO-PRO-1 as described above.
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Results |
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We have previously shown that infection with HSV-1 inhibits activation of caspases 3 and 8 during Fas- or UV-induced apoptosis (13). However, it has become increasingly clear that multiple interrelated caspases are involved in the process of apoptosis. Caspase 9 is the predominant target for cleavage and activation after mitochondrial transmembrane potential collapse through the release of cytochrome c (15). Caspase 6, an effector caspase, appears to be closely linked with the nuclear morphological changes seen in apoptosis, probably through the cleavage of nuclear lamins (16). We therefore investigated the inhibitory effect of HSV-1 on activation of these caspases and the role of Us5 in their inhibition.
To obtain a broad view of caspase inhibition by HSV, we first extended
our previous work with HSV to evaluate the inhibition of caspases 6 and
9. Similar to our findings with caspases 3 and 8, infection with the
HSV-1 laboratory strain E115 inhibited activation of caspases 6 and 9
after apoptosis induction by UV or anti-Fas Ab (Fig. 1
A). In agreement with our
previous results, the inhibition of the effector caspase (caspase 6,
40–55% inhibition) was more profound that the inhibition of the
initiator caspase (caspase 9, 30–45% inhibition). Similar inhibition
of caspase 6 and 9 activation was seen after infection with any of
three low passage clinical isolates of HSV-1 (data not shown). In
contrast to the inhibition seen with HSV-1, HSV-2 laboratory strain 333
did not show inhibition of caspase activation under the conditions of
this study (Fig. 1B), nor did either of two HSV-2 clinical
isolates tested (data not shown).
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The deletion virus experiments of the previous section demonstrate
that Us5 is required for maximal HSV-1 inhibition of the
infected cell’s apoptotic machinery. Other groups have reported
similar experiments showing requirements for Us3, ICP22, and
ICP27 (17, 18, 19, 20). However, none of these studies has
demonstrated that the protein products of these have direct
anti-apoptotic activity, and it is possible that deletion of these
genes results in altered expression or activity of other viral
anti-apoptosis genes. To investigate whether the Us5
gene product, gJ, has direct anti-apoptotic activity, we expressed
Us5/gJ in Jurkat cells using a Tet-regulated system.
Expression of Us5 and its inducibility by Tet were confirmed
by RT-PCR (Fig. 3A).
Transfected cells with Us5/gJ expression induced were
strongly protected from Fas- or UV-induced apoptosis, in contrast to
untransfected control cells or cells with Us5/gJ expression
uninduced (Fig. 3, B and C). In agreement with
our previous data using deletion viruses, inhibition of apoptosis
ranged from about 40 to 60% after treatment with anti-Fas Ab or UV
radiation. To confirm visual inspection of the cells, we performed flow
cytometry using a combination of PI and YO-PRO-1. Permeability to
YO-PRO-1 is an early event in apoptosis (21) and occurs
well before loss of membrane integrity and permeability to PI. Cells
expressing Us5/gJ were refractory to cell shrinkage after
apoptosis induction, as determined by forward scatter (data not shown).
In addition, the Us5/gJ-expressing cells were resistant to
the entry of YO-PRO-1 after UV or anti-Fas relative to control
Us5/gJ OFF cells (Fig. 3D).
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A). Inhibition of caspases
was confirmed by incubating Us5/gJ-induced or -uninduced
cells with the cell-permeable polycaspase substrate D2R
(14), followed by flow cytometry (Fig. 4B). The
degree of caspase inhibition in the Us5/gJ transfectants
(40–70%) was similar to that seen in earlier experiments using
deletion and rescue virus. However, it is important to recognize
that transfection with a single gene, such as Us5, can lead
to protein overexpression relative to levels seen in viral infection.
Thus, these results do not imply that Us5/gJ accounts for
the full anti-apoptotic effect of HSV, nor do they conflict with
previous work showing the involvement of other HSV genes.
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40% of cells showing
an apoptotic low membrane potential (Fig. 5). In contrast, transfectants with
Us5/gJ expression induced (Us5 ON) showed
markedly less mitochondrial potential loss (only 10% with low
potential) after either stimuli, suggesting that the Us5
gene product also interferes with this process.
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The data presented above suggest that HSV-1 in general and
Us5/gJ in particular can mediate broad inhibition of caspase
activation. Because the CTL granule component grB is related to
the caspases in terms of substrate specificity, and because grB
acts to cleave and thus activate caspases, we investigated whether
HSV-1 inhibited grB-induced caspase activation. To do this, we made
mitochondria-free extracts from HSV-1-infected or control uninfected
Jurkat cells, then added purified grB and measured caspase 3 activation
at intervals thereafter. Lysates from HSV-1-infected cells showed that
caspase 3 activation was inhibited relative to control uninfected
lysates (Fig. 6A). To
investigate the role of Us5/gJ in this inhibition, we
performed similar experiments with lysates from Jurkat cells infected
with Us5 deletion or rescue virus. The Us5
deletion and the rescue virus showed equivalent inhibition of
grB-induced caspase activation, similar to that seen with wild-type
HSV-1 strain E115 (Fig. 6B). Surprisingly, however,
transfectants with Us5/gJ expression induced (Us5
ON) were strongly protected from grB-induced caspase activation (Fig. 6C), suggesting that Us5/gJ is sufficient to
inhibit grB-induced caspase activation. In the setting of viral
infection; however, other viral gene products can perform this function
in the absence of Us5/gJ. To determine whether
Us5/gJ could protect intact cells from grB-induced death, we
delivered grB into cells using sublytic levels of purified perforin.
Whereas cells with Us5/gJ expression turned off were highly
susceptible to killing by perforin/grB, transfectants with
Us5/gJ expression induced were markedly protected from
perforin/grB-induced apoptosis (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which the Us5/gJ inhibits apoptosis remains unclear. gJ is a small glycoprotein that can be found on the surface of infected cells (23). Other than its anti-apoptotic role, no function for this protein is known, and viruses deleted for Us5 have been reported to be phenotypically normal in culture, with apparently normal plaque formation and cell-to-cell spread of the virus (24, 25). We have not detected homology between Us5 and other known viral or cellular anti-apoptosis genes. Because gJ is localized to the cell membrane, we have suggested that it might act upon signaling via the Fas receptor or antagonize activation of caspase 8. Caspase 8 is known to be involved in amplification of the activation of other caspases (26), which might explain the general dampening of activation of multiple caspases by Us5/gJ. However, the ability of Us5/gJ to inhibit cell death induced by perforin plus grB argues against this mechanism, because caspase inhibitors do not prevent perforin/grB-induced death (27, 28). The data may be more compatible with a function of Us5/gJ at the mitochondria, which would also manifest as general dampening of caspase activation during apoptosis, especially in type II cells such as Jurkat (29). Further studies are clearly needed to establish the mechanism of action of Us5/gJ.
It has been suggested that the granule-mediated mechanisms of apoptosis induction, such as grB, evolved to bypass the effects of inhibitors of receptor-mediated caspase activation, and it appears that the granule-mediated mechanisms constitute the main pathway used by HSV-specific CTL (8). After engagement of a target cell by CTL and granule exocytosis, grB is delivered to the target cell cytoplasm and nucleus in a perforin-dependent process (30, 31, 32). grB, a serine protease, can cleave multiple substrates in the target cell (33, 34, 35, 36, 37), and triggers apoptosis. Activation of caspases is required for grB-induced nuclear DNA fragmentation, but not for the induction of cell death by grB, and it is clear that inhibition of caspases alone is insufficient to fully protect cells from grB (27, 28). One mechanism by which grB may initiate apoptosis is via the cleavage of Bid, which leads, in turn, to mitochondrial disruption (38, 39, 40). grB has also been reported to cleave some cell death substrates directly, such as inhibitor of caspase-activated DNase (ICAD) (40, 41). Cleavage of ICAD leads to release of caspase-activated DNase, resulting in DNA fragmentation of the target cell. However, other groups have reported that inhibition of caspases prevents grB-induced DNA fragmentation (28, 42, 43, 44). The reasons for these discrepancies remain unclear, but grB is less efficient than caspase 3 at the cleavage of ICAD (41), and it is likely that caspase activation amplifies the direct activity of grB. We have previously shown that HSV-1 infection protects cells from DNA fragmentation induced by CD4+ CTL (6), which would be consistent with our observation that HSV-1 infection inhibits caspase activation.
Even in systems where caspase inhibition prevents grB-induced DNA fragmentation, target cell lysis remains unaffected (28). Inhibition of caspases together with blockade of mitochondria result in protection of cells from grB-induced lysis, but even in this case whole CTL granules still induce target cell death in a grB-independent process (45). The observation that various aspects of cell death can be regulated and inhibited separately leads to the question of exactly what aspects of cell death or apoptosis pose a threat to successful viral replication. We have shown that HSV-1 infection protects cells from DNA fragmentation, and yet membrane exposure of phosphatidylserine during apoptosis is not inhibited (6). These observations suggest that DNA fragmentation or a similarly regulated process poses a threat to HSV, whereas phosphatidylserine exposure does not. It is possible that phosphatidylserine exposure by infected cells is even advantageous to the virus, by promoting phagocytosis of infected cells and, thus, cell-to-cell spread of the virus.
In addition to grB, other granule components, such as grA, can induce cell death, although perhaps with slower kinetics of action that grB. The grA pathway appears to be independent from and complementary to the grB pathway (10, 11), and these pathways may represent redundancy built into the immune system to counter viral escape mechanisms. Additional experiments will be necessary to assess the effects of other granule components, such as grA, on HSV-infected cells, and whether HSV gene products modulate the action of the other granule components.
Finally, it is important that studies on immune killing of
virus-infected cells look beyond single measures of cytotoxicity and
consider the range of cellular changes collectively known as cell death
or apoptosis. The different regulation of DNA fragmentation and cell
lysis emphasizes this point, and it remains unclear which, if either,
of these processes most closely correlates with efficient control of
viral replication. Furthermore, a prelytic halt of HSV replication has
been described by CTL (46), and it was recently shown that
CD8+ CTL can control viral gene expression and
reactivation from neurons without lysis of these cells
(47). This effect was suggested to be mediated through the
action of IFN-
. grA may have noncytotoxic mechanisms of action in
addition to its ability to induce apoptosis, and grA appears to
restrict interneuronal spread of HSV in ganglia of experimentally
infected mice without inducing neuronal cytotoxicity (48).
These findings suggest that nonlytic (or noncytotoxic) mechanisms are
also important in the control of viral infections. Thus, the control of
viral infections may be based on a combination of cytokine-mediated,
receptor-mediated, and lytic granule-mediated mechanisms. The ultimate
yardstick by which the effectiveness of these must be measured is the
degree to which viral replication is prevented. Thus, the uneasy truce
between HSV and the host immune response may be even more complicated
than previously appreciated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Keith R. Jerome, D3-100 Fred Hutchinson Cancer Research Center, Seattle, WA 98104. E-mail address: kjerome{at}fhcrc.org
3 Abbreviations used in this paper: gr, granzyme; gJ, glycoprotein J; PI, propidium iodide; ICAD, inhibitor of caspase-activated DNase.
Received for publication December 7, 2000. Accepted for publication July 20, 2001.
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irradiation. J. Immunol. 161:3936.This article has been cited by other articles:
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