(Received for publication, July 6, 1995; and in revised form, August 8, 1995)
From the
Cytotoxic T-lymphocytes (CTLs), by virtue of their ability to
recognize and induce apoptotic death of virus-infected cells, comprise
a major antiviral defense mechanism. The induction of apoptosis by CTLs
can be completely accounted for by two mechanisms: (i) a
Ca-dependent component that involves the exocytotic
release of serine proteases known as granzymes from CTL granules and
their subsequent insertion into the target cell to induce apoptosis and
(ii) a Ca
-independent component that involves the
activation of Fas, a receptor on the target cell membrane that triggers
apoptosis. Although viruses have evolved several indirect mechanisms
for evading the CTL response, direct inhibition of the apoptotic
cascade has never been described. We now show for the first time that
the cowpox virus protein CrmA, a protease inhibitor of the serpin
family, is capable of inhibiting CTL-mediated cytolysis. The inhibitory
effect is largely the result of blockade of the
Ca
-independent (i.e. Fas-mediated) component
of CTL killing. CrmA thus represents the first example of a viral gene
product capable of directly blocking CTL-mediated cell death.
Cytotoxic T-lymphocytes (CTLs) ()represent a major
anti-viral mechanism, as they kill virally infected cells by inducing
apoptosis(1) . Recent studies have demonstrated that killing by
CTLs is completely accounted for by two mechanisms: (i) the insertion
of serine proteases known as granzymes, and in particular the
Asp-specific protease Granzyme B, into the target
cell(2, 3) , which potently activates the cell's
endogenous death pathway and (ii) activation of the Fas antigen, an
apoptosis-inducing receptor on the target cell
surface(2, 3, 4, 5, 6, 7) .
The granzyme pathway requires extracellular Ca
, as it
involves the exocytotic release of granzymes, along with the
pore-forming protein perforin, from granules in the CTL (reviewed in (8) ). The Fas pathway, on the other hand, is
Ca
-independent(7) , requiring only the
interaction between Fas ligand expressed on the CTL surface and Fas
present on the target cell membrane. Experimental verification of the
existence of these two pathways comes from analysis of mice carrying
germline inactivation of the Granzyme B gene(9) , from
knockouts of perforin (2, 3) (which is necessary for
the insertion of granzymes into the target cells), and from mice
carrying inactivating mutations in either the Fas ligand or Fas
genes(2, 3) .
Many viruses have evolved indirect
mechanisms to evade the host CTL response to
infection(10, 11) . These include altering expression
of cell surface major histocompatibility proteins and altering the
expression of specific viral antigens to avoid CTL
recognition(10) . Until now, however, no example of a viral
evasive mechanism that blocks CTL killing by directly interfering with
the apoptotic cascade has been described(10) . CrmA is a cowpox
virus gene product that was originally identified as a serpin that
inhibits the interleukin-1 converting enzyme
(ICE)(12, 13) , which proteolytically processes
pro-interleukin-1
to yield mature, active
interleukin-1
(14, 15) . ICE, like Granzyme B, is
an Asp-specific protease(14, 15) , and CrmA, as a
member of the serpin family of protease inhibitors, inhibits ICE by
forming an active site-directed complex(12, 13) . With
the discovery that ICE and ICE-like proteases may function in
apoptosis(16, 17, 18, 19, 20, 21, 22) it
seemed plausible that CrmA might have anti-apoptotic activity in
addition to its ability to inhibit the processing of
interleukin-1
. This was confirmed in a variety of model systems,
including nerve growth factor withdrawal-(23) , serum
withdrawal-(18) , tumor necrosis factor-, and Fas-induced
apoptosis(21, 24, 25) .
In particular, the
finding that CrmA can block Fas-induced apoptosis (21) suggested to us that it might function as an inhibitor of
CTL-mediated killing, since Fas is one of the two effector pathways
utilized in this process. Furthermore, a recent report demonstrated
that, at least in vitro, Granzyme B, an essential component of
the Ca-dependent cytolysis pathway, is inhibited by
CrmA(26) , suggesting that CrmA may be capable of attenuating
both CTL killing pathways. We directly tested this hypothesis in a
CTL-mediated cytotoxicity assay using target cells stably transfected
either with a vector control, with a CrmA expression construct, or with
an inactive point mutant CrmA expression construct. We show here that
CrmA functions to inhibit CTL killing and that it is a more potent
inhibitor of the Ca
-independent (i.e. Fas-based) mechanism than of the Ca
-dependent (i.e. granzyme-based) component of CTL killing. These studies
identify CrmA as the first example of a viral gene product that
directly blocks the lethal apoptotic cascade induced by CTLs and
suggest that CrmA may function to allow virally infected cells to
combat immune surveillance.
NK cell cytotoxicity assays were carried out as described above except that the cells were not stimulated and there was no PHA present during the assay.
To determine whether CrmA can function to inhibit the lethal
cascade induced upon interaction of the CTL with its target, we
utilized BJAB cells (a Fas-expressing human B-cell line) stably
transfected with either vector control, CrmA, or inactive point mutant
CrmA expression constructs (21, 27) as target cells in
a 24-h PHA-facilitated allogeneic CTL-mediated cytolysis assay based on Cr release from target cells. We chose to examine
Cr release after 24 h because we anticipated that both
Ca
-dependent and Ca
-independent
cytotoxicity would occur at this time point. Two vector-transfected
clones (V1 and V4), two CrmA-transfected clones (CrmA 2 and CrmA 3),
and two mutant CrmA-expressing clones (mutant CrmA 12 and mutant CrmA
17) were selected that have been previously described and characterized (21, 27) . Mutant CrmA carries a Thr
Arg point
mutation at codon 291, a critical site in the reactive site loop of
CrmA. This mutation abolishes protease-inhibitory capacity without
significantly altering tertiary structure, as measured by transverse
urea gradient gel electrophoresis(21) . This mutation has also
been shown to abolish the ability of CrmA to inhibit tumor necrosis
factor- and Fas-induced cell death(21) .
When examined for
susceptibility to CTL-mediated cytolysis, the parental BJAB cell line
was killed effectively (data not shown), as were vector transfected
cells (Fig. 1A, clones V1 and V4), in a dose-dependent
fashion over a range of killer:target cell ratios. Lines expressing
CrmA, however, were significantly protected from CTL-mediated killing (Fig. 1A, clones CrmA 2 and CrmA 3). When spontaneous
background release of Cr from BJAB lines incubated for 24
h without the addition of CTLs was assessed, there was no significant
difference between vector and CrmA lines (data not shown). Thus, the
difference in susceptibility to killing between vector and
CrmA-expressing lines in the CTL-mediated cytolysis assays is not
simply due to a decreased intrinsic propensity of the CrmA lines to
undergo spontaneous lysis.
Figure 1:
Wild-type CrmA, but
not mutant CrmA, inhibits CTL-mediated cytolysis. A, clonal
lines of BJAB cells stably transfected with either a vector control
(clones V1 () and V4 (
)) or CrmA expression construct
(clones CrmA 2 (
) and CrmA 3 (
)) were used as target cells
in a 24-h PHA-facilitated allogeneic CTL-mediated cytolysis assay based
on
Cr release as described under ``Experimental
Procedures.'' B, clonal lines of BJAB cells stably
transfected with either a vector control (clone V1 (
)), CrmA
(clone CrmA 2 (
)), or mutant CrmA (clones mutant CrmA 12
(
) and mutant CrmA 17 (
)) expression constructs were
similarly loaded with
Cr and analyzed in the 24-h
CTL-mediated cytolysis assay. Each of the data points shown in both A and B represents the mean of samples run in
triplicate, and the standard deviations were always less than 5% of the
mean. Each experiment was independently repeated at least three times
with similar results. Although absolute values of chromium release
cannot be directly compared between experiments due to variation
arising from differences in blood donors, variation in the degree of
protection by CrmA relative to vector or mutant CrmA was less than 5%.
100% chromium release corresponded to values ranging between 4000 and
12,000 cpm, depending upon the individual
experiment.
To determine whether the ability of CrmA to inhibit CTL-mediated cytolysis required its protease-inhibitory capacity, we examined cell lines that expressed comparable levels of the point mutant of CrmA, that, as mentioned above, lacks protease-inhibitory activity(21) . When tested in the same cytotoxicity assay, both lines expressing mutant CrmA were as sensitive to CTL-mediated cytolysis as a vector transfectant, whereas wild-type CrmA was clearly protective (Fig. 1B).
Because
killing by CTLs is the result of activation of both granzyme and Fas
pathways, it was of interest to determine whether one or both of these
was modulated by CrmA, since previous studies indicated that CrmA could
potentially inhibit both pathways(26, 27) . To
investigate this, we took advantage of the Ca dependence of granzyme-mediated killing and the Ca
independence of the Fas pathway. The calcium chelator, EGTA, can
be used to block granule exocytosis(30, 31) , since
calcium is necessary for granzyme release from the CTL. Thus, one can
selectively assess Ca
-independent killing. In the
absence of EGTA, CrmA was protective compared with vector controls, but
some residual cytotoxicity was present (Fig. 2A). In
the presence of EGTA, however, CrmA was totally protective (Fig. 2B), indicating that CrmA can completely block
the Ca
-independent component of CTL killing. These
data suggest that the residual killing observed in the CrmA-expressing
lines in the absence of EGTA (Fig. 2A) was exclusively
due to the Ca
-dependent granzyme pathway and that at
least under the experimental conditions employed, CrmA was ineffective
at blocking this pathway.
Figure 2:
CrmA completely blocks the
Ca-independent component of CTL-mediated killing. A, BJAB cells stably transfected with either vector (clones V1
(
) and V4 (
)) or CrmA (clones CrmA 2 (
) and CrmA 3
(
)) expression constructs were analyzed in the 24-h CTL-mediated
cytolysis assay in the absence of EGTA as described under
``Experimental Procedures.'' B, the same cell lines
were analyzed in the 24-h CTL-mediated cytolysis assay in the presence
of 10 mM EGTA and supplemented with 4 mM Mg
, as described under ``Experimental
Procedures.'' Each of the data points shown in both A and B represents the mean of samples run in triplicate, and the
standard deviations were always less than 5% of the mean. Each
experiment was independently repeated at least three times with similar
results. Although absolute values of chromium release cannot be
compared between experiments due to variation arising from differences
in blood donors, variation in the degree of protection by CrmA relative
to vector was less than 5%. 100% chromium release corresponded to
values ranging between 4000 and 12,000 cpm, depending upon the
individual experiment.
Studies of Granzyme B-knockout mice (9) have suggested that Cr release in the first 4
h after CTL-target interaction is likely to involve the granzyme-based
pathway only. We therefore carried out a series of experiments using a
4-h cytolysis assay in place of the 24-h assay employed for the
experiments described above. In this 4-h assay there were no
significant differences in CTL sensitivity of vector-transfected and
CrmA-transfected target cells (Fig. 3A). Furthermore,
EGTA abolished all cytotoxicity in the 4-h assay (Fig. 3B), confirming that the cytolysis at this time
point was entirely due to the Ca
-dependent mechanism
and indicating that CrmA therefore does not afford protection from the
Ca
-dependent pathway. We also found that CrmA did not
confer any protection from death induced by NK cells (data not shown).
This result is consistent with the hypothesis that CrmA does not block
the Ca
-dependent component of cytotoxicity, since
NK-mediated cytolysis appears to be entirely accounted for by the
degranulation pathway(32) .
Figure 3:
CrmA does not block the
Ca-dependent component of CTL-mediated cytolysis. A, BJAB cells stably transfected with either vector (clones V1
and V4) or CrmA (clones CrmA 2 and CrmA 3) expression constructs were
analyzed in a 4-h CTL-mediated cytolysis assay in the absence of EGTA
as described under ``Experimental Procedures.'' B,
the same cell lines were analyzed in a 4-h CTL-mediated cytolysis assay
except in the presence of 10 mM EGTA and supplemented with 4
mM Mg
, as described under
``Experimental Procedures.'' Each of the data points shown in
both A and B represents the mean of samples run in
triplicate, and the standard deviations were always less than 5% of the
mean. Each experiment was independently repeated at least three times
with similar results. Although absolute values of chromium release
cannot be compared between experiments due to variation arising from
differences in blood donors, variation in the degree of protection by
CrmA relative to vector was less than 5%. 100% chromium release
corresponded to values ranging between 4000 and 12,000 cpm, depending
upon the individual experiment.
Whereas the 4-h Cr
release assay measured only Ca
-dependent cytolysis,
we suspected that assessing DNA fragmentation might be a more sensitive
measure of total apoptosis at this early time point, as it
precedes membrane lysis in the case of Fas-mediated apoptosis. When
both vector control and CrmA-transfected lines were examined for
induction of DNA fragmentation by CTLs, CrmA-expressing lines showed
significantly less DNA fragmentation (Fig. 4), consistent with
protection from early DNA fragmentation induced by Fas ligation.
Figure 4:
CrmA blocks CTL-mediated DNA
fragmentation. BJAB cells stably transfected with either vector (clones
V1 () and V4 (
)) or CrmA (clones CrmA 2 (
) and CrmA 3
(
)) expression constructs were labeled with
[methyl-
H]thymidine and DNA
fragmentation induced by a 4-h incubation with CTLs as described under
``Experimental Procedures.'' Each data point represents the
mean of samples run in triplicate, and the standard deviations were
always less than 5% of the mean. Two independent experiments yielded
similar results, and the degree of protection by CrmA relative to
vector varied by less than 5% between experiments. 100% DNA
fragmentation corresponded to absolute values between 400 and 1000 cpm
depending upon the individual experiment.
Many animal cells undergo apoptosis when exposed to viruses, a response likely to have evolved as a means to halt the production of new viral particles. Not surprisingly, then, the ability to inhibit apoptosis is a recurring theme in viral pathogenesis. Viruses are under tremendous evolutionary pressure to evolve mechanisms that will inhibit apoptotic death of the infected host cell, allowing them time to replicate. The Epstein-Barr (33) and African swine fever viruses(34) , for example, encode genes that resemble bcl-2, a mammalian gene capable of inhibiting apoptosis. Interestingly, while these genes may offer protection from a multitude of apoptosis-inducing insults, they have not been shown to protect from CTL killing.
The importance of CTL-mediated killing in the host-virus interaction is suggested by the diversity of viral mechanisms that have evolved to combat T cell-mediated lysis. Prior studies of viral anti-CTL defenses have identified mechanisms of evasion of recognition by CTLs (reviewed in (10) ) by such means as down-regulation of host surface molecules required for CTL recognition or modification of viral antigens. CrmA represents the first example of a viral protein that blocks CTL-mediated killing by a direct effect on the lytic pathway. Koup (10) has previously suggested, on theoretical grounds, the existence of a class of viral proteins that inhibit the CTL-initiated lethal apoptotic cascade. We now suggest that CrmA may function in such a capacity and thus may be the first member of such a class of viral proteins.
CrmA is a gene
product encoded by cowpox virus and although it is not required for in vitro replication, it facilitates high titer viral
replication in vivo(35) . It was previously thought
that CrmA achieved this by inhibiting ICE, the enzyme responsible for
the conversion of interleukin-1 from its inactive precursor to the
active mature molecule(12) . Such an inhibition would dampen
the host inflammatory response to the viral infection. While this may
still indeed be the case, the fact that CrmA partially inhibits CTL
killing supports the contention that CrmA might allow the virus more
time to replicate by attenuating the CTL-based host defense mechanism.
Our findings suggest that the Ca-independent
component of the CTL mechanism is targeted by CrmA. These data are
consistent with the previous finding that Fas-induced apoptosis
triggered by agonist monoclonal antibodies is blocked by expression of
CrmA(27) . The lack of blockade of the granzyme-based pathway
is of importance, since Granzyme B is inhibited by CrmA in vitro(26) and since, based on gene knockout studies in mice,
Granzyme B has been shown to play a nonredundant role in CTL
killing(9) . The inability of CrmA to protect from
Ca
-dependent killing might be explained by the
finding that CrmA is by 2 orders of magnitude a poorer inhibitor of
Granzyme B than it is of ICE(26) . Thus, it is possible that
higher levels of CrmA expression than those achieved in the present
studies may be needed to detect effects on Granzyme B-mediated
apoptosis. Since ICE and ICE-like molecules appear to play a role in
Fas-mediated killing, this may explain the greater propensity of CrmA
to inhibit the Ca
-independent component of the
apoptotic mechanism. The fact that CrmA does block
Ca
-independent CTL killing, however, raises the
possibility that the Fas-based pathway may play a greater role in
eliminating virus-infected cells than has been previously appreciated,
since cowpox virus appears to have evolved a mechanism to combat it.