(Received for publication, February 3, 1997, and in revised form, February 18, 1997)
From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602 and the § Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
Molluscum contagiosum virus proteins MC159 and
MC160 and the equine herpesvirus 2 protein E8 share substantial
homology to the death effector domain present in the adaptor molecule
Fas-associated death domain protein (FADD) and the initiating death
protease FADD-like interleukin-1-converting enzyme (FLICE)
(caspase-8). FADD and FLICE participate in generating the death signal
from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95
receptor. The flow of death signals from TNFR-1 occurs through the
adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by
overexpression of the downstream adaptors TRADD and FADD. Neither viral
molecule, however, inhibited FLICE-induced killing, consistent with an
inhibitory action upstream of the active death protease. These data
suggest the existence of a novel strategy employed by viruses to
attenuate host immune killing mechanisms. Given that bovine herpesvirus
4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13
also possess significant homology to the viral inhibitory molecules
MC159, MC160, and E8, it may be that this class of proteins is used
ubiquitously by viruses to evade host defense.
Cell suicide is a defense mechanism employed by host cells to inhibit viral replication and persistence. As a consequence, viruses have evolved numerous strategies to attenuate apoptosis (1). For example, the Epstein-Barr virus (EBV) encodes BHRF1, a homolog of the mammalian anti-apoptosis molecule bcl-2, and the cowpox virus encodes a serpin-like protein, CrmA, that blocks apoptosis by inhibiting proteases belonging to the caspase family.
Molluscum contagiosum virus (MCV) is the only poxvirus family member
still associated with human disease (2). It usually causes asymptomatic
cutaneous neoplasms that can spontaneously regress. However, with the
advent of immunocompromised populations, particularly those afflicted
with acquired immunodeficiency syndrome, MCV infection has become a
clinical challenge (3). Unfortunately, due to the inability to grow the
virus in tissue culture cells and the lack of a suitable animal model,
little is known about host-virus relationships (4). Equine herpesvirus
2 (EHV2) is a member of the -herpesvirus subfamily that also
includes herpesvirus saimiri, EBV, Kaposi's sarcoma-associated
herpesvirus (KSHV), and bovine herpesvirus 4 (5, 6). Although EHV2 is
ubiquitously distributed and has been implicated as a pathogen in
immunosuppressed states, its mode of evading the host immune response
is uncertain. However, the recent availability of the MCV and EHV2
genome sequences has begun to identify genes that suggest potential
pathogenic mechanisms (7, 8).
MCV, surprisingly, does not encode many of the immunoregulatory molecules present in other poxviruses, especially those that antagonize the host cytokine-mediated inflammatory response. These include CrmA and a soluble TNFR-like1 molecule (7). In contrast, EHV2 encodes an interleukin-10-like factor that may attenuate the host immune response (8). Regardless, MCV and EHV2 do not encode previously identified inhibitors of apoptosis (1). Instead, MCV and EHV2 encode novel members of an emerging family of molecules characterized by the presence of a death effector domain (DED) originally identified in signaling molecules engaged by the death receptors TNFR-1 and CD-95 (7, 8).
Both TNFR-1 and CD-95 contain a stretch of approximately 60-80 amino acids within their cytoplasmic domains termed the death domain. Upon activation the receptor death domains bind to corresponding death domains within the adaptor molecules TRADD (for TNFR-1) and FADD (for CD-95) (9-12). Utilizing the same mechanism, TRADD is able in turn to recruit FADD to the TNFR-1 signaling complex (13). FADD appears to play a central role as a conduit for death signals from both receptors as dominant negative versions that retain the death domain but lack the amino-terminal segment effectively attenuate both TNFR-1- and CD-95-induced killing (14). Since it is likely that the amino-terminal domain of FADD functions to engage downstream components of the death pathway, it has been termed the DED (14). The importance of this domain was dramatically underscored by the discovery of its presence within the prodomain of the death protease FLICE (15-17). It appears that the DED of FADD binds to the corresponding DED motif within the FLICE prodomain and thereby recruits this death protease to the receptor signaling complex. Therefore, a homophilic binding mechanism involving DEDs is responsible for assembly of the receptor death signaling complex. Disruption of such a complex by DED-containing viral gene products could potentially abrogate propagation of the death signal.
Human embryonic kidney 293, 293T, and 293-EBNA cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, nonessential amino acids, L-glutamine, and penicillin/streptomycin. Mammalian expression vectors encoding TNFR-1, CD-95, FADD, FLICE, MC159, MC160, E8, and CrmA were cloned into pcDNA3 (Invitrogen). The expression vector for TRADD was kindly provided by Dr. David Goeddel (Tularik, Inc.).
In Vitro Binding AssayFull-length FADD and truncated N-FADD were expressed as GST-fusion proteins as described previously (18). [35S]Methionine-radiolabeled MC159 was obtained by in vitro transcription/translation using the TNT T7-coupled reticulocyte lysate system (Promega). Binding reactions were performed as described previously (18).
Transfection, Coimmunoprecipitation, and Western AnalysisTransient transfections of 293T cells were performed as described previously (19). Cells were harvested 40 h following transfection, immunoprecipitated with the indicated antibodies, and analyzed by immunoblotting.
Cell Death AssayFor CD-95, TRADD, and FLICE killing,
experiments were performed in 293-EBNA cells and in 293 cells for
TNFR-1 and FADD killing. cDNAs encoding putative apoptosis inducers
(0.5-0.8 µg) and potential inhibitors (2.5 µg) were cotransfected
in each experiment together with the reporter plasmid pCMV
-galactosidase. Cells were fixed and stained 24-30 h following
transfection. The percentage of apoptotic cells was determined by
calculating the fraction of round membrane-blebbed blue cells as a
function of total blue cells. All assays were evaluated in duplicate
and the mean and standard deviation calculated.
MCV encodes two
closely related proteins: MC159 and MC160 (6). The NH2
termini of the 241-amino acid protein MC159 and the 371-amino acid
protein MC160 contain two motifs homologous to DEDs present at the
NH2 terminus of FADD and repeated in tandem within the
prodomain of FLICE (7). Interestingly, the DED motif is also present
within EHV2-encoded protein E8 (171 amino acids), Kaposi's
sarcoma-associated herpesvirus-encoded protein K13 (139 amino acids),
and bovine herpesvirus 4-encoded protein E1.1 (182 amino acids) (Fig.
1) (20, 21). Unlike the MCV proteins, full-length E8,
K13, and E1.1 encode only DED motifs. K13 encodes one complete and one
incomplete DED, whereas E8 and E1.1 encode two DEDs. Each DED of these
viral proteins contains a highly conserved module RXDL/I(L)
(X is any amino acid) that is also conserved in the DEDs of
FADD and FLICE. It appears that many other herpesviruses also encode
DED-like molecules. Examples include the herpesvirus saimiri protein
VG71 and human herpesvirus 6 protein U15 (22, 23).
E8 and MC159 Inhibit TNFR-1- and CD-95-induced Apoptosis
The presence of DEDs within E8, MC159, and MC160
suggests that these viral proteins might potentially antagonize the
FADD-FLICE interaction and thereby attenuate TNFR-1- and CD-95-mediated
apoptosis. Indeed, overexpression of MC159 significantly inhibited
TNFR-1- and CD-95-induced cell death (Fig.
2A). The degree of inhibition was
substantially greater than that achieved with the catalytically inactive dominant-negative version of FLICE (data not shown) and comparable in potency with CrmA. MC160 also inhibited TNFR-1- and
CD-95-induced cell death (Fig. 2B), as did E8 (Fig.
2C).
E8 and MC159 Inhibit TRADD and FADD Killing but Not FLICE Killing
Additional studies were undertaken to delineate the point
at which MC159 and E8 were exerting their inhibitory effect on the TNFR-1- and CD-95-induced death pathways. As shown in Fig.
3, both MC159 and E8 significantly blocked both TRADD
and FADD killing, suggesting that these inhibitors must function
downstream of these adaptor molecules. In contrast, MC159 and E8 did
not inhibit FLICE-induced death, suggesting that they must act upstream
of active FLICE. The overexpression of FLICE zymogen results in
autoactivation to the active protease that is potently inhibited by the
viral serpin CrmA (Fig. 3C).
MC159 Binds FADD, whereas E8 Binds FLICE
Binding studies were
undertaken to investigate the potential mechanism utilized by E8 and
MC159 to attenuate TNFR-1- and CD-95-induced cell death (Fig.
4). Radiolabeled in vitro translated MC159
was precipitated with various GST-fusion proteins immobilized onto glutathione-Sepharose beads, including GST-FADD, GST-NFADD containing only the NH2-terminal DED (amino acid residues: 1-82), or
GST alone (Fig. 4A). As expected from the homophilic binding
nature of DEDs, MC159 strongly bound GST-FADD and GST-NFADD, but not GST alone.
To demonstrate the association of the viral inhibitory molecules with FADD or FLICE in vivo, 293 cells were transiently transfected with expression constructs encoding epitope-tagged versions of the respective molecules (Fig. 4). Consistent with the in vitro binding results, MC159 precipitated with FADD (Fig. 4B), but not with FLICE (data not shown). Conversely, E8 strongly associated with FLICE (Fig. 4C), but not with FADD (data not shown). This binding specificity of MC159 and E8 suggested that distinct mechanisms were employed by these two inhibitors. MC159 binds to FADD and presumably blocks its interaction with FLICE. The reverse is probably true for E8 in that it binds FLICE and inhibits its interaction with FADD. However, when FLICE is overexpressed (upon transfection), the binding of E8 is unable to overcome the propensity of this caspase to autoactivate (Fig. 3C). Therefore, once FLICE is active, E8 has no inhibitory influence. Regardless, either mechanism would disrupt the assembly of the receptor·FADD· FLICE signaling complex and abrogate activation of downstream caspases. Further studies will be needed to substantiate these proposed mechanisms.
We thank Dr. Andrew Davison for providing the EHV2 DNA; Arul Chinnaiyan, Marta Muzio, James Pan, and Karen O'Rourke for providing reagents and helpful discussions; and Ian Jones for his expertise in preparing the figures.