Vol. 16, No. 19, pp. 2465-2478, October 1, 2002
REVIEW
Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection
Andrea
Cuconati,1 and
Eileen
White1,2,3,4
1 Howard Hughes Medical Institute, Center for Advanced
Biotechnology and Medicine, 2 Department of Molecular Biology
and Biochemistry, Rutgers University, and 3 Cancer Institute
of New Jersey, Piscataway, New Jersey 08854, USA
 |
Introduction |
Cellular BCL-2-related proteins (cBCL-2s) function as regulators of
programmed cell death (apoptosis), in part by
modulating the release of proapoptotic signaling molecules from
mitochondria. These factors act to promote activation of cysteine
proteases of the caspase family and thereby propagate signaling of
apoptotic cell death. DNA viruses are known to encode homologs of
cellular antiapoptotic BCL-2 proteins (vBCL-2s), and the role of
vBCL-2s in various aspects of viral infection and the mechanism by
which they function have been gradually emerging. It is now apparent that inhibition of apoptosis by vBCL-2s in infected cells can prevent
premature death of the host cell, which would impair virus production;
can enable efficient emergence from latency; can facilitate persistent
infection; and contributes to the avoidance of immune surveillance by
the host. Thus, apoptosis is clearly a mechanism used by the host
immune system and the infected host cell itself as part of the
antiviral response. Deregulation of this delicate host-pathogen
interaction can alter the course of virus replication, which may
explain aspects of viral disease. Recent evidence suggests that vBCL-2s
may target the core cellular proapoptotic machinery for inhibition,
perhaps to secure a broad spectrum of apoptosis inhibition to the
infected cell. Furthermore, because vBCL-2 family members and some of
their cellular counterparts are oncogenic, deciphering their mode of
action may be useful in understanding and thwarting human cancer. In
this review we outline the role and the mechanism of action of vBCL-2
proteins in infected cells and how and why their function may be
distinct from some of their cellular homologs.
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Identification of vBCL-2s |
The first indication that viruses encode BCL-2 functional homologs
and that their manipulation of apoptosis is a requirement for normal
productive infection came from observations made with adenovirus-infected cells. One of the oncogenes of adenovirus, E1B 19K, was shown to be required to prevent the degradation
of host-cell and viral DNA and enhanced cytopathic effects during productive virus infection (Pilder et al. 1984
; Subramanian et al.
1984; Takemori et al. 1984; White et al. 1984). These activities bore a
striking resemblance to apoptosis and suggested that the E1B 19K
protein may function as an apoptosis inhibitor in productively infected
human cells (White et al. 1991). At that time, BCL-2 was known to
function by promoting cell survival, which was the first link to the
genetic pathway of apoptosis regulation in mammalian cells (Vaux et al.
1988). We now know that the function of antiapoptotic cBCL-2s is
required for normal development, and an inappropriate gain-of-function
mutation is associated with promotion of human cancer. BCL-2 is the
prototypical member of a family of proteins that act to regulate
apoptosis in a process that is evolutionarily conserved from humans to
flies and nematodes (Gross et al. 1999a). That viruses should have
stolen or copied this activity to serve their own ends should not be surprising.
E1B 19K and BCL-2 can functionally substitute for one another in the
suppression of apoptosis during virus infection and oncogenic transformation, indicating that viruses did, indeed, see fit to encode
inhibitors of apoptosis (Rao et al. 1992; White et al. 1992; Tarodi et
al. 1993; Chiou et al. 1994b; Subramanian et al. 1995). Although the
sequence homology between E1B 19K and BCL-2 is extremely weak, the most
conserved residues in the dozen or so viral serotypes in which the
sequence of E1B 19K is known are also conserved in BCL-2 (Tarodi et al.
1993; Chiou et al. 1994b). The presence of both sequence and functional
homology between E1B 19K and BCL-2 indicates that E1B 19K is an
adenovirus BCL-2 homolog. Furthermore, the E1B 19K protein also blocks
apoptosis induced by a wide variety of stimuli even outside the context of virus infection, indicating that it is functioning at a regulatory point shared by many apoptotic pathways (White 2001). The task then was
to determine why adenoviruses encoded an antiapoptotic gene product,
and how it worked. A prediction was that if inhibition of apoptosis
were important for the life cycle of one virus, that it might be an
important activity of other viruses. Indeed, the 
-herpesvirus
Epstein-Barr virus (EBV) is recognized to encode a viral homolog of
BCL-2, BHRF1 (Cleary et al. 1986; Henderson et al. 1993). It is now
known that not only adenoviruses, but also other -herpesviruses
(Rabizadeh et al. 1993; Russo et al. 1996; Nicholas et al. 1997; Virgin
et al. 1997; Afonso et al. 2001; Rivailler et al. 2002), and the
pox-related viruses fowlpox (FPV; Afonso et al. 2000) and African swine
fever virus (ASFV; Afonso et al. 1996; Brun et al. 1996) all encode
vBCL-2s. These viruses additionally encode alternate, non-BCL-2-type,
partially redundant functions to block apoptosis, which underscores the importance of apoptosis inhibition during viral infection (Roulston et
al. 1999). These non-BCL-2-type antiapoptotic functions are apparently
sufficient for disabling apoptosis by many other viruses that lack
vBCL-2s. We can only conclude that regulating apoptosis during
infection is a high priority for many viruses, and one recurring theme
is to use a BCL-2-like mechanism to do so.
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Virus infection and a need to encode vBCL-2s |
Infection in vitro with proapoptotic mutant adenoviruses that lack
E1B 19K function is associated with impaired virus production, indicating that it might be advantageous to the virus to disable apoptosis to facilitate replication during productive infection. This
may be necessary because virus-dependent deregulation of cell cycle
control initiates an act of altruistic cell suicide on the part of the
individual infected cell, which the virus may benefit from inhibiting
to sustain host cell viability to enable viruses to maximally
replicate. As the immune response to viral infection includes multiple
mechanisms for induction of apoptosis of infected cells, inhibition of
apoptosis could allow virus replication even when infected cells are
under immune attack, thereby contributing to the repertoire of viral
immune stealthing functions. Furthermore, inhibition of apoptosis may
enable viruses to establish latency, or facilitate the emergence from
latency and the establishment of a chronic, persistent infection. Swift
and efficient induction of apoptosis upon infection could restrict the
host range of virus replication, or facilitate rapid virus release and
cell-to-cell spread of virus. Finally, an altered apoptotic state of
the infected host cell itself, owing to disease or cell type, could
promote or diminish virus replication. How and when apoptosis
regulation is important to a virus may depend on the specific aspects
of the virus life cycle. In this review, we discuss what is known about
vBCL-2 function in adenoviruses, herpesviruses, and poxviruses.
| |
Adenoviruses |
The Adenoviridae constitutes a large family of small DNA
tumor viruses that have mammal and bird hosts (Horwitz 2001; Shenk 2001). Human adenoviruses are pathogens, some of which are the cause of
as much as 10% of all childhood pneumonias, whereas others cause
conjunctivitis and outbreaks of acute respiratory disease. Of the human
adenoviruses, there are 51 different serotypes that mainly infect and
replicate in epithelial cells of the respiratory and gastrointestinal
tract, where they cause mild to severe disease in immunocompetent
individuals that can become fatal in those who are immunocompromised.
Following the initial acute infection, adenovirus is shed for many
months after individuals are symptomatic, suggesting that the virus can
establish a persistent infection. Adenovirus encodes numerous immune
stealthing functions, including several antiapoptotic activities that
may facilitate the establishment of a persistent infection. There is
also evidence of the presence of adenoviral DNA without infectious
virus in human tissues, particularly lymphocytes, which is indicative
of latent infection. Further evidence of persistent or latent
adenovirus infections comes from immunosuppressed individuals, who
apparently become reinfected, often fatally, from an endogenous source
or from a donor organ. Unfortunately, there is no animal model that
faithfully reproduces adenovirus disease in humans, although infection
of cotton rats or mice that are semi- or nonpermissive for virus
replication can recapitulate some of the aspects of human adenovirus disease.
Productive adenovirus infection of the human epithelial cell line HeLa,
which is a model system for productive replication, begins with virus
entry and early gene expression. E1A expression, in
particular, is required to stimulate the transcription of the other
viral early genes, and to create an S-phase-like environment for viral
DNA replication to proceed. Adenovirus gene products are typically
multifunctional, often by having multiple binding sites for the
cellular proteins whose activities they modulate. In this way, an
adenovirus can retain a fairly small genome (35 kb) without encoding
direct sequence homologs of cellular genes. However, the E1B 19K
protein may be an exception, as it appears to be a stripped-down,
minimal version of cBCL-2s. There are, however, distinct differences
between E1B 19K and other vBCL-2s and cBCL-2s.
The product of the adenovirus E1A oncogene deregulates the
normal cellular constraints of cell cycle regulation by interacting with the retinoblastoma protein (RB), its relatives p107 and p130, and
the transcriptional coactivators p300 and CBP. These activities of E1A
also activate apoptosis that is inhibited by expression of the products
of the E1B gene. E1B 55K, in cooperation with E4 ORF6,
interacts with and promotes the degradation of p53 in the proteasome
(Querido et al. 2001), whereas the E1B 19K protein blocks apoptosis
induced by E1A and also that signaled by death receptor ligands such as
tumor necrosis factor-
(TNF-), Fas ligand (FasL), and tumor
necrosis factor--related apoptosis-inducing ligand (TRAIL). Death
receptor signaling represents part of an antiviral immune response in
the infected host. The adenovirus E3 gene products also inhibit
apoptosis induced by death receptor signaling, but this function is
dispensable for productive virus replication in vitro (Wold et al.
1999). Viral DNA replication provides the switch to viral late-gene
expression, the assembly and accumulation of virus particles in the
nucleus (Shenk 2001), and eventual release of virus from the cell by a
mechanism facilitated by expression of the adenovirus death protein
(ADP; Tollefson et al. 1996). Rodent cells, which are semi- or
nonpermissive for virus replication, are additionally susceptible to
transformation by the adenovirus oncogenes E1A and
E1B, expressed from either plasmids or upon infection with
adenovirus. Deregulation of cell growth control by E1A activates
p53-dependent apoptosis that is directly inhibited by the E1B 55K
protein, and blocked downstream of p53 by vBCL-2, the E1B 19K protein
(Debbas and White 1993; Sabbatini et al. 1995; Henry et al. 2002).
Thus, coordinate stimulation of cell cycle progression and disabling of
apoptosis are required for both oncogenic transformation and productive
infection. These apoptosis-related virus-host interactions also impact
the use of adenovirus as a vector for human gene therapy (Vorburger and Hunt 2002), and as replication-based anticancer agents (McCormick 2001). This increases the importance of establishing a detailed understanding of the interaction between adenovirus and its human host,
in which apoptosis regulation plays a major role.
| |
Herpesviruses |
The Herpesviridae is a large family of DNA viruses (Kieff
and Rickinson 2001; Roizman and Pellett 2001), representing more than
130 herpesviruses that are highly species-specific, with hosts that
range from mammals and birds to amphibians and reptiles. There are
three subfamilies of herpesviruses: , which includes herpes simplex
virus (HSV) and varicella zoster virus (VZV); 
, which includes
cytomegalovirus (CMV); and , which includes human herpes virus 8 (HHV8) and Epstein-Barr virus (EBV). There are nine herpesviruses that
have humans as the natural host, and these viruses are associated with
significant disease. Herpesviruses productively replicate, and as with
adenoviruses, viral DNA replication and virion assembly occur in the
host nucleus. This leads to the ultimate destruction of the infected
cell, but the infected host usually lives. Virus entry is followed by
immediate early and early gene expression, viral DNA replication,
partial late and late gene expression and synthesis of viral structural
proteins, and virion assembly. The comparably large genome of
herpesviruses (125-200 kb) accommodates a substantial repertoire of
genes involved in nucleic acid metabolism to create an ideal
environment for virus replication, as well as still more gene products
involved in disabling the antiviral defense system of the cell and host.
Herpesviruses also undergo latency and reactivation cycles, dictated by
the environment of the host cell, that are far better understood
mechanistically than those of adenoviruses (Kieff and Rickinson 2001;
Roizman and Pellett 2001). Many herpesviruses have specific genes
required for the establishment of latency, which occurs in the natural
host and in specific cell types. In the latent state, the viral genome
is present as a circular episome that replicates along with the DNA of
the host cell, and there is no infectious virus produced. The latent
state, however, does permit virus replication upon reactivation.
Herpesviruses are also associated with the occurrence of some human
cancers, which may represent an aberrant manifestation of the virus
life cycle, particularly latency. The most notorious cancer-associated
human herpesviruses are the -herpesviruses EBV and HHV8. EBV plays a
major role in the development of Burkitt's lymphoma (BL), Hodgkin's Disease (HD), and nasopharyngeal carcinoma (Rickinson and Kieff 2001),
and HHV8 is associated with the development of Kaposi's sarcoma and
primary effusion lymphomas (Moore and Chang 2001). Tumorigenesis caused
by herpesviruses, in general, appears to result from a combination of
restricted expression of viral genes and genetic changes to the host
cell, exemplified by c-myc translocation to the immunoglobulin
heavy chain locus in cases of BL. The tumorigenic potential of
herpesviruses is often enhanced in the immunocompromised, indicating an
active role of the immune system in suppressing cancer development.
Unlike adenoviruses, herpesviruses have many host-acquired genes (Kieff
and Rickinson 2001; Moore and Chang 2001; Roizman and Pellett 2001).
The predominantly lymphotrophic -herpesviruses encode vBCL-2s, and
as many as a dozen homologs of other cellular genes, and many of these
genes have been modified compared with their cellular counterparts. The
viral D-type cyclin (vCYCLIN), for example, does not respond to normal
regulation by the cell, as may also be the case for the vBCL-2s.
Although the well-known - (HSV and VZV) and - (CMV) herpesviruses
do not encode an obvious vBCL-2 homolog, they do encode other genes
with antiapoptotic functions. Among the -herpesviruses that do
encode vBCL-2s, there is substantial evidence that they function as
apoptosis inhibitors in response to diverse stimuli. EBV BHRF1 blocks
apoptosis induced by death receptor signaling (Kawanishi 1997), growth
factor withdrawal (Henderson et al. 1993; Foghsgaard and Jäättelä
1997), granzyme B (Davis et al. 2000), irradiation,
chemotherapeutic drugs (McCarthy et al. 1996; Khanim et al. 1997),
deregulated c-myc (Fanidi et al. 1998), and p53 (Tarodi et al.
1994; Theodorakis et al. 1996). A second EBV vBCL-2 homolog, BALF1, may
either block apoptosis (Marshall et al. 1999) or antagonize BHRF1
(Bellows et al. 2002). HHV8 vBCL-2 inhibits apoptosis induced by BAX or
vCYCLIN overexpression or Sindbis virus infection (Cheng et al. 1997b;
Sarid et al. 1997; Ojala et al. 1999). Herpesvirus saimiri (HVS) vBCL-2
blocks FAS-, dexamethasone-, Sindbis virus-, and DNA damage-mediated
apoptosis (Nava et al. 1997; Derfuss et al. 1998). Herpesvirus papio
BHRF1 blocks apoptosis induced by DNA damage (Meseda et al. 2000), and murine -herpesvirus 68 (HV68) blocks apoptosis by anti-FAS antibody and TNF- (Wang et al. 1999). A logical assumption is that the function of -herpesvirus vBCL-2s is necessary to protect the infected cell from apoptosis induction by either the immune system or
by the infected cell itself. Perhaps vCYCLIN can stimulate cell cycle
progression and apoptosis in a manner analogous to that of adenovirus
E1A, thereby necessitating the need for expression of a vBCL-2.
EBV BHRF1 appears to be dispensable for lytic infection in vitro
(Marchini et al. 1991; Lee and Yates 1992), but given the repertoire of
other antiapoptotic genes encoded by EBV, particularly the gene
encoding latent membrane protein 1 (lmp1; Kieff and Rickinson 2001), this is not surprising. Until recently, it has not been clear
what the physiological role of vBCL-2 was in the life cycle of
herpesvirus, which was in part owing to the lack of a good animal model
for herpesvirus infection that faithfully represented productive acute,
latent, and reemergent infection. The EBV- and HHV8-related murine
-herpesvirus HV68, and the corresponding mutant viruses that lack
v-cyclin and vbcl-2, have enabled some of these
issues to be addressed.
| |
Poxviruses |
The Poxviridae are large, structurally complex viruses with
DNA genomes that comprise some of the most dreaded human and animal pathogens yet discovered. Poxviruses are composed of two groups that
infect either vertebrates (Chordopoxvirinae) or arthropods (Entimopoxvirinae; Moss 2001). In the life cycle of
vaccinia, considered as the prototypical poxvirus, entry and
uncoating are followed by immediate transcription of viral genes by
a set of transcription factors that is contained within the virion
particle itself. Replication of the viral DNA through the activity
of a virus-encoded DNA polymerase occurs in the cytoplasm, a feature that is shared by African swine fever virus (ASFV). In the later stages of infection, expression of structural proteins culminates in
the assembly and release of daughter virion particles (Moss 2001). The
large genome of poxviruses (130-300 kb) encodes numerous non-BCL-2
apoptosis inhibitors such as the caspase inhibitory serpins or viral
FLICE inhibitory proteins (vFLIPs), as well as soluble TNF receptors
(Barry and McFadden 1998; Roulston et al. 1999), which may obviate the
need to encode a vBCL-2. Despite the large repertoire of antiapoptotic
and immune stealthing functions encoded by poxviruses, vBCL-2s are
encoded by FPV and the poxvirus-like ASFV. The role that apoptosis
plays in the pathology and the life cycle of poxviruses is not well
understood, but it almost certainly functions as a factor in limiting
viral replication, given the extensive array of antiapoptotic functions
encoded by this virus family. As such, antiviral therapies that
stimulate apoptosis of the infected cell may prove useful in
controlling poxvirus infections.
It is unclear exactly how infection by poxviruses induces apoptosis,
but it may be a generalized response to subversion of host-cell
metabolism such as the shutoff of host macromolecule synthesis that
occurs during viral infection. Fowlpox (FPV) is capable of inducing
hyperplasia in infected tissues (Cheevers et al. 1968), and this effect
may represent a loss of cell cycle control to which the cell responds
with a death signal. FPV is a member of the genus
Avipoxvirus, and is the only poxvirus known to encode a BCL-2
homolog (FPV039), which closely resembles the prosurvival BCL-2 family
member MCL-1 (Afonso et al. 2000). It is possible that other members of
the same genus will also turn out to encode vBCL-2s.
ASFV is a large enveloped DNA virus encoding 151 predicted genes.
Although it is the only representative of the new family Asfarviridae, ASFV shares some characteristics with the
poxviruses, namely, the structure of its genome and cytoplasmic
localization of its replication complexes. African swine fever is an
economically important, often fatal illness of domestic pigs that is
transmitted by ticks. The affected tissues are destroyed by apoptosis
(Gomez-Villamandos et al. 1995; Ramiro-Ibanez et al. 1996; Oura et al.
1998), and the degree of cell death induction correlates with the
lethality of the infecting strain. However, the tissues displaying the
highest proportion of apoptotic cells also have the lowest rates of
viral replication (Oura et al. 1998), a phenomenon that complements the
observation that ASFV-induced apoptosis requires only adsorption and
uncoating, and not viral replication (Carracosa et al. 2002). Although
the pathology of ASFV disease is typified by the massive induction of
apoptosis, the virus also expresses apoptotic inhibitors. Therefore,
how or why cell death occurs is unclear, but it may be a result of
"bystander" apoptosis induced in uninfected tissues (Oura et al.
1998).
ASFV encodes numerous cell-acquired genes, including the BCL-2 homolog
A179L in the BA71V strain (originally termed LMW5-HL in the Malawi
strain; Neilan et al. 1993; Yanez et al. 1995). When exogenously
expressed, A179L can protect cells from apoptosis induced by IL-3
withdrawal (Afonso et al. 1996), the interferon-induced p68 kinase
(PKR; Brun et al. 1996), and the inhibition of macromolecular synthesis
(Revilla et al. 1997). Interestingly, A179L can also repress
baculovirus-induced apoptosis in Sf9 insect cells (Brun et al. 1998),
indicating a very low degree of species-specificity, as would be
required of a viral protein that probably must function in both a
mammal and an arthropod. The role of the ASFV vBCL-2 in the virus life
cycle and its mechanism of action remain to be determined.
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Functional and structural organization of the BCL-2 family |
The cBCL-2 family encodes more than a dozen mammalian members in
addition to homologs in Caenorhabditis elegans and
Drosophila melanogaster (Gross et al. 1999a). Family members
are highly conserved throughout evolution, most notably in highly
conserved BCL-2 homology regions (BH) 1-4. Three different subgroups
of BCL-2-related proteins have been identified: those that promote
survival such as BCL-2 and BCL-xL, which encode BH1-BH4;
those that promote cell death such as BAX and BAK, which encode
BH1-BH3; and those that promote cell death such as BID, NBK/BIK, and
BAD, which encode only BH3 (BH3-only proteins). BH3 is a binding domain
used for homo- and heterodimerization between BCL-2 family members. A
large region in the center of the BCL-2 proteins encompassing BH1-BH3
forms a hydrophobic pocket that serves as the receptor for BH3. BH4, which is only present within the antiapoptotic family members, is
thought to be a regulatory domain. Most family members also possess a
putative membrane-targeting transmembrane region (TM) at the C terminus.
BCL-2 family members function by interacting with each other, and by
either promoting or antagonizing the function of the binding partner
(Gross et al. 1999a). These protein-protein interactions rely on the
BH3 of one protein interacting with the hydrophobic cleft created by
BH1-BH3 of the other (Sattler et al. 1997). The BH3-only protein BAD,
for example, can interact with BCL-xL to antagonize its
survival function. Alternatively, the BH3-only protein BID can be
cleaved by caspase-8 during death receptor-mediated apoptosis to
truncated BID (tBID), which reveals its BH3 and allows interaction with
BAX and BAK to promote their proapoptotic function (Gross et al.
1999a). In turn, BCL-2 and BCL-xL interact with BID and tBID
to prevent BAX and BAK activation, blocking apoptotic signaling. BCL-2
and BCL-xL can also bind BAX and BAK, and block apoptosis.
However, the preferred targets of BCL-2 and BCL-xL may depend
on differential BH3-binding affinity and the physiological context.
BH3-only proteins are upstream regulators of BAX and BAK that promote
apoptosis by facilitating the release of mitochondrial proteins such as
holo-cytochrome c and SMAC/DIABLO from the intermembrane space
(Cheng et al. 2001; Wei et al. 2001; Zong et al. 2001; Degenhardt et
al. 2002). Cytochrome c is a cofactor for APAF-1-dependent caspase-9 activation in the apoptosome complex, whereas SMAC/DIABLO is
an antagonist of the inhibitor of apoptosis proteins (IAPs) that act as
direct negative regulators of caspase activation (Wang 2001; Shi 2002).
Apparently, many death signaling pathways rely on BH3-only proteins
activating BAX and BAK to propagate caspase activation and apoptosis
through mitochondria, as cells deficient for both BAX and BAK are
resistant to apoptosis mediated by BH3-only proteins and their upstream
stimuli (Lindsten et al. 2000; Cheng et al. 2001; Wei et al. 2001; Zong
et al. 2001; Degenhardt et al. 2002). Without BAX and BAK, the release
of cytochrome c, SMAC/DIABLO, and perhaps other proapoptotic
mitochondrial proteins is severely impaired, illustrating the
functional requirement for BAX and BAK for this activity.
Interestingly, cells singly deficient in either BAX or BAK are capable
of releasing cytochrome c, activating caspase-9, and
undergoing apoptosis, indicating that BAX and BAK are functionally
redundant. Thus, abrogation of apoptotic signaling may require
inhibition of the upstream BH3-only protein or inactivation of both BAX
and BAK.
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Interaction with and inhibition of proapoptotic BAX and BAK
by vBCL-2s |
The first indication that the adenovirus vBCL-2 E1B 19K protein
functioned biochemically by similar mechanisms to cBCL-2s emerged when
yeast two-hybrid screening using E1B 19K as bait identified BAX and BAK
as interacting proteins (Farrow et al. 1995; Han et al. 1996a). BAX was
originally cloned as a protein that coimmunoprecipitated from mammalian
cells with BCL-2 (Oltvai et al. 1993), and BAK was isolated from
two-hybrid screens using BCL-2 as bait (Chittenden et al. 1995; Kiefer
et al. 1995). E1B 19K inhibited BAX- and BAK-mediated apoptosis
similarly to BCL-2, and it did so by interacting with the BH3 of BAX
(Han et al. 1996a), and probably also that of BAK. In the case of
BAX-mediated apoptosis, BAX BH3 was both necessary and sufficient for
E1B 19K interaction, and E1B 19K BH1 mutants that lose BAX-binding
activity no longer block apoptosis (Han et al. 1996a; Han et al.
1998a). In an analogous manner, BH1 mutations in ASFV vBCL-2 (A179L),
or BH1 and BH2 mutations in EBV BHRF1, also eliminate their
antiapoptotic activity (Khanim et al. 1997; Revilla et al. 1997). This
suggests that E1B 19K, and perhaps other vBCL-2s, act as receptors for
BH3s of proapoptotic BAX and BAK, and antagonize their function.
Activation of the proapoptotic function of BAX and BAK requires
interaction with BH3-only proteins and a series of changes in BAX and
BAK conformation, and the E1B 19K-BAX and E1B 19K-BAK interactions
were informative in establishing the order of events during the
activation process. E1B 19K-BAX interaction requires a change in BAX
conformation provided, for example, by hit-and-run binding of tBID to
BAX, presumably to expose BAX BH3. Thus the E1B 19K protein is not
normally found bound to BAX in healthy cells in the absence of a death
stimulus (Perez and White 2000; Sundararajan and White 2001;
Sundararajan et al. 2001; Cuconati et al. 2002). This requirement for
altered protein conformation to reveal binding activity of
proapoptotic proteins may explain some conflicting reports on
vBCL-2-BAX and vBCL-2-BAK binding activity. HHV8 vBCL-2 does not bind
in vitro translated BAX and BAK (Cheng et al. 1997b), but the purified
vBCL-2 protein does bind BAX and BAK BH3 peptides with high affinity
(Huang et al. 2002). EBV BALF1 associates with BAX and BAK (Marshall et
al. 1999), or rather may antagonize BHRF1 (Bellows et al. 2002).
Indeed, other vBCL-2s may interact with and inhibit BAX and BAK. EBV
BHRF1 binds BAK, BCL-2, BCL-xL, and BCL-xS, but not
BAX (Theodorakis et al. 1996); and HVS vBCL-2 binds BAX and BAK (Nava
et al. 1997). Because vBCL-2 proteins may interact with cellular
apoptotic regulators in a conformation-dependent fashion, it may be
essential to examine their function and binding abilities in vivo and
in the presence of a physiologically relevant death stimulus.
Furthermore, as conformational changes in BAX and BAK as well as
dimerization of BCL-2 family members are artifactually induced by
detergents commonly used for immunoprecipitation (Hsu and Youle 1997,
1998), it is important to examine BCL-2 family protein interactions in the absence of these detergents.
| |
vBCL-2s may prefer BAX and BAK as targets over BH3-only proteins |
Two-hybrid screening with the E1B 19K protein as bait identified the
first proapoptotic BH3-only protein NBK/BIK as well as BNIP3 (Boyd et
al. 1994, 1995; Han et al. 1996b), revealing that BAX and BAK were not
necessarily the only vBCL-2 targets. Although E1B 19K interacts with
and inhibits apoptosis induced by NBK/BIK and BNIP3 overexpression, the
physiological context of these activities still remains to be
determined. Although BCL-2 also interacts with NBK/BIK, E1B 19K does
not interact with the BCL-2 and BCL-xL interacting BH3-only
proteins BAD, BID, and tBID (Chen et al. 1996; Perez and White 2000).
Thus, there are distinct binding specificities that distinguish E1B
19K, and possibly other vBCL-2s, from cBCL-2s. Perhaps vBCL-2s would
prefer to target for inhibition the core apoptotic machinery,
represented by BAX and BAK, rather than the upstream regulators
represented by the BH3-only proteins. A possible preference of vBCL-2s
for BAX and BAK over BH3-only proteins may be a matter of efficiency,
as BH3-only proteins may vary in their regulation and expression in
different cell types. This may make them more elusive, and thereby less
desirable targets for vBCL-2s.
| |
Sequence and structural similarities between vBCL-2 and
cBCL-2 proteins |
vBCL-2 proteins predominantly act as apoptosis inhibitors in cases
in which their function has been examined, and they encode most of the
recognizable BH regions (Figs. 1,
2). Thus,
vBCL-2s are expected to be somewhat similar in structure to cBCL-2s.
BH3 and BH4 are least conserved at the level of sequence identity among
vBCL-2s, and between vBCL-2s and cBCL-2s, but the structure in these
regions may nonetheless be conserved (see below). Many, but not all,
vBCL-2s also possess a predicted C-terminal TM region (Figs. 1, 2). The
adenovirus E1B 19K protein does not possess a TM, and may be
membrane-associated by virtue of posttranslational fatty acid acylation
(McGlade et al. 1987). All vBCL-2s have a recognizable BH1 and at least
one other BH2 or BH3, likely preserving the sequence of the highly
conserved central part of the proteins, which can serve as a receptor
for BH3. BAX BH3 is sufficient for interaction with the central highly
conserved region of E1B 19K that encompasses BH3 and BH1; therefore,
the absence of a recognizable BH2 still enables formation of the
presumptive BH3-binding site (Han et al. 1996a, 1998a).
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Figure 1.
Sequence and structural homology among vBCL-2s.
Multiple sequence alignment among vBCL-2 proteins encoded by
adenoviruses, herpesviruses, and poxviruses. The coding regions for the
vBCL-2 genes were subjected to multiple sequence alignment (Megalign,
DNA*, Clustal Method). Regions with conserved, identical amino acids
are shaded in yellow. The green overlay indicates the location of
-helices from the solution structure of HHV8 vBCL-2 (Huang et al.
2002). The locations of the BCL-2 homology (BH) and transmembrane (TM)
regions are indicated in red. GenBank accession numbers for the protein
sequences used are listed below in parentheses. (HHV8vbcl-2) Human
herpesvirus 8 vBCL-2 (NP-572068); (HVSorf16) herpesvirus saimiri orf16
vBCL-2 (NP-040218); (ASFV5HL) African swine fever virus A179L/LMW-5HL
vBCL-2 (Q07818); (FPOX) fowlpox virus 039 vBCL-2 (NP-039002); (HV68M11)
herpesvirus 68 M11 vBCL-2 (NP-044912); (EBVBHRF1) Epstein-Barr virus
BHRF1 vBCL-2 (P03182); (AD2E1B19K) adenovirus type 2 E1B 19K vBCL-2
(NP-040510); (AD9E1B19K) adenovirus type 9 E1B 19K vBCL-2 (AAD16304).
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Figure 2.
Conservation of BH and TM regions among vBCL-2s.
BCL-2 homology regions and their conservation among viral homologs.
Predicted domain organization of vBCL-2 proteins. Indicated is the
probable presence of the BCL-2 homology (BH) and transmembrane (TM)
regions based on sequence homology (Fig. 1). Note that low homology,
particularly in BH4, BH3, and BH2, suggests the absence of these
regions in some vBCL-2s, which could nonetheless be conserved at the
level of protein structure. (HHV8 vBCL-2) Human herpesvirus 8 vBCL-2;
(HVS orf16) herpesvirus saimiri open reading frame 16 vBCL-2; (ASFV
A179L) African swine fever virus A179L/LMW-5HL vBCL-2; (FPV 039)
fowlpox virus 039 vBCL-2; (HV68 M11) herpesvirus 68 M11 vBCL-2; (EBV
BHRF1) Epstein-Barr virus BHRF1 vBCL-2; (AD2E1B19K) adenovirus type 2 E1B 19K vBCL-2.
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With the determination of the crystal structure of BCL-xL
minus the TM, it emerged that BCL-2 family members bore a striking similarity to the pore-forming domains of bacterial toxins (Muchmore et
al. 1996; Aritomi et al. 1997). BCL-xL possesses two
central helices surrounded by amphipathic helices interconnected
sometimes by large unstructured loops. The structure of BCL-2 is highly similar to that of BCL-xL (Petros et al. 2001), and,
surprisingly, so is that of the BH3-only protein BID (Chou et al. 1999;
McDonnell et al. 1999). Thus structural homology is conserved between
BID and BCL-xL even in the absence of obvious sequence
homology outside of BH3. BID cleavage to tBID is predicted to reveal
its BH3 and thereby the proapoptotic activity of tBID. The arrangement
of the helices in BCL-xL and BCL-2 creates a hydrophobic
cleft on the surface of the proteins that serves as a binding pocket
for BH3 regions (Sattler et al. 1997). The solution structure of BAX with its transmembrane region intact revealed that the TM resided within the hydrophobic cleft reserved for BH3 (Suzuki et al. 2000). This suggested that homo- and heterodimerization between BCL-2 family
members requires a change in protein conformation whereby the TM would
be displaced from the BH3-binding pocket to allow protein interactions
to take place. These protein interactions could occur by BH3 or TM
binding of one BCL-2 family member to the pocket of another. Because
the TM of BAX resides in the BH1-BH3-binding pocket for BH3 and
presumably occludes binding to the pocket (Suzuki et al. 2000), the
absence of TM in E1B 19K raises the possibility that its BH3-binding
pocket is constitutively exposed. If so, then the E1B 19K protein, and
perhaps other vBCL-2s that lack a TM, may be active scavengers of
exposed BH3s. The overall theme of changes in protein conformation to
control binding and function is reminiscent of how the pore-forming
domains of bacterial toxins work, and this method may have been usurped
by cBCL-2s and exploited by vBCL-2.
When activated, the pore-forming domains of bacterial toxins undergo a
change in conformation that exposes the two central helices, which are
then thought to insert into the bacterial membrane and oligomerize to
form a pore to release bacterial contents (Gazit et al. 1998; Gilbert
et al. 1999; Manoj and Aronson 1999; Gerber and Shai 2000; Wallace et
al. 2000). The structural homology of BCL-2 family members with the
domains of bacterial toxins suggests the possibility of a related
biochemical activity. Although it is still controversial how BCL-2
family members function at the biochemical level, there are distinct
parallels between the function of BAX and BAK and that of the toxin
pore-forming domains.
The solution structure of the first vBCL-2, that of HHV8 vBCL-2, has
recently been determined. The overall fold is highly similar to that of
the cBCL-2s, with the exception that the unstructured loop regions
interconnecting the helices are smaller (Huang et al. 2002). The
vBCL-2s are uniformly smaller proteins than their cellular
counterparts, and the preservation of the overall fold at the expense
of the unstructured loops may have allowed that to occur (Figs. 1, 2).
In cBCL-2s the loops encode sites for phosphorylation and proteolysis,
which may be required for regulating the function of these proteins in
development and maintaining homeostasis (Haldar et al. 1995; Uhlmann et
al. 1996; Chang et al. 1997; Cheng et al. 1997a; Bellows et al. 2000).
These regulatory events, which are uniformly inhibitory to
antiapoptotic family members, would be expected to be detrimental to
the viral counterparts, which may account for the absence of
conservation of the loops. In contrast to cBCL-2s, E1B 19K is not
functionally altered by phosphorylation (McGlade et al. 1989), and
vBCl-2s are not cleaved (Bellows et al. 2000). Structural similarity is
preserved, however, even with limited conservation of amino acid
sequence. For example, BH4/1 and BH3/2, which are poorly
conserved in HHV8, correspond to 1 and 2, and BH4 and BH3 of
BCL-xL and BCL-2 (Huang et al. 2002). Whether the poor
conservation of primary sequence in these regions in other vBCL-2s
still permits conservation of protein structure, or represents distinct
differences in structure and function, remains to be determined.
The hydrophobic BH3-binding cleft is conserved in HHV8 vBCL-2, and it
binds with high affinity to peptides encoding BH3 derived from BAK and
BAX, and, to a much lesser extent, BAD (Huang et al. 2002). This was
somewhat surprising because HHV8 vBCL-2 was originally thought not to
bind BAK and BAX (Cheng et al. 1997b). A BH3-only protein-dependent
change in conformation of BAX and BAK that dislodges TM and reveals BH3
and the hydrophobic cleft, may be required for HHV8 vBCL-2 binding
potential. This may necessitate that BAX and BAK binding activity be
assessed in vivo in the presence of a physiological death stimulus.
Taken together, these observations suggest that vBCL-2 proteins may
target BAX and BAK, and may not be subject to the regulatory mechanisms
that control the function of, particularly inactivate, cBCL-2s.
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BAX and BAK function as an antiviral response to abort virus
replication and persistent infection |
In adenovirus-infected cells, the expression of E1A causes the
inappropriate entry of the cell into an S-phase-like state, which
facilitates replication of the viral genomic DNA (White 1994, 2001).
The resultant onset of apoptosis is thought to compromise the viability
of the infected cell, and possibly abort the viral replication cycle.
To counteract this effect, expression of E1B 19K functions to block
apoptosis induced by cell cycle deregulation. Deletion or mutation of
the E1B 19K gene confers the cyt and deg viral phenotypes, in which the infected cell's genomic DNA as well as
replicated viral DNA are degraded (Pilder et al. 1984; Subramanian et
al. 1984; Takemori et al. 1984; White et al. 1984). The E1B 19K viral
mutants usually replicate to lower levels than the wild-type virus,
presumably because of apoptotic destruction of the infected cell. The
replication of E1B 19K mutants can be rescued by the inhibition of
apoptosis in the infected cell (Chiou and White 1998; Cuconati et al.
2002). These observations support the concept of apoptosis as a
limiting factor in the life cycle of DNA viruses, to which some viruses
respond with the expression of antiapoptotic homologs of BCL-2.
The antiapoptotic function of E1B 19K can mostly be attributed to its
ability to bind to BAX and BAK. In productively infected cells, E1A
expression correlates with a conformational change in BAK in which the
N terminus is exposed (Cuconati et al. 2002). In cells infected with
E1B 19K viral deletion mutants, the altered form of BAK forms a complex
with BAX. This BAK-BAX interaction may be a key event, because it is
followed by stepwise conformational changes in BAX that likely result
in exposure of the BH3, mirroring the effects of tBID interaction with
BAX. BAK and BAX form high-molecular-weight complexes in apoptotic
cells (Antonsson et al. 2000; Eskes et al. 2000; Korsmeyer et al. 2000;
Sundararajan and White 2001; Sundararajan et al. 2001). These BAX and
BAK complexes are associated with the release of cytochrome c
and SMAC/DIABLO from the intermembrane space of mitochondria into the
cytosol, resulting in the cytochrome c-dependent activation of
the APAF1/caspase-9 complex, and presumably SMAC/DIABLO-dependent
inhibition of IAPs, which may facilitate activation of caspase-3. In
adenovirus-infected cells, E1B 19K binds BAK and abrogates the
interaction of BAK and BAX, which prevents the conformational changes
that activate BAX and the release of cytochrome c (Cuconati et
al. 2002). Therefore, the mechanism by which E1B 19K blocks apoptosis
to allow the progress of productive infection centers on a block of the
mitochondrial checkpoint caused by inhibition of BAK and BAX.
Although E1B 19K may possess functions independent of influencing
BAX/BAK interactions (Rao et al. 1997; Han et al. 1998b; Kasof et al.
1998; Perez and White 1998), studies of adenovirus infection of cells
deficient for BAX and BAK demonstrated that this is a major aspect of
E1B 19K activity in apoptosis regulation. The dependence of
adenovirus-induced apoptosis on BAK and BAX is demonstrated by the
observation that in semipermissive mouse cells deficient for BAX and
BAK, the growth disadvantage that is conferred by an E1B 19K deletion
is abolished (Cuconati et al. 2002). Therefore, the loss of a vBCL-2
can be complemented by the absence of BAK and BAX. Given the striking
difference in viral replication in the BAX- and BAK-deficient
semipermissive mouse cells, it will be of interest to examine the
replication capacity of adenoviruses in permissive human cells
deficient for BAX and BAK.
If cells deficient for BAK and BAX are infected with adenovirus, the
onset of cytopathic effects is delayed, and the absence of BAK and BAX
allows more efficient viral replication than that which occurs in
infection of wild-type cells. This may be responsible for the
propensity of these cells to allow long-term persistent infection to
occur in culture (A. Cuconati and E. White, in prep.). BCL-2
overexpression facilitates persistent infection by human immunodeficiency virus (HIV) and Sindbis virus (Levine et al. 1993;
Antoni et al. 1995), indicating that the course of virus infection can
be altered by either the gain of a survival activity or the loss of an
apoptotic activity. Persistent infection by adenovirus has been
observed repeatedly both in vitro and in clinical cases. Primary
cultures of human monocytes, continuous B-cell lines, and isolated
lymphoid tissues have been documented as sustaining long-term
adenoviral infections (Van Der Veen and Lambriex 1973; Chu et al. 1992;
Flomenberg et al. 1996). The variables that may determine whether
persistent infection occurs may relate to a particular genotype of the
infecting viral strain, the expression levels of proapoptotic factors
in the affected tissues, or to the insensitivity of those tissues to
E1A expression. It is unknown whether the establishment of persistent
infection in those cases depends on an inability of the infected
tissues to initiate an apoptotic response, but it arises as an
intriguing possibility. In experimental or clinical situations in which
persistent infection is observed, analysis of the apoptotic status of
the infected cell type may well reveal that such cells are not
responding with the cell death signaling cascade, suggesting that
therapies meant to stimulate the progress of cell death may aid in the
management of persistent adenovirus infections in the clinic.
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vBCL-2s enable emergence from latency and establishment of chronic,
persistent infection |
As the coordination between deregulation of cell cycle control and
inhibition of apoptosis by E1A and E1B 19K is critical for productive
replication and persistent infection in vitro by adenoviruses, these
same activities appear to be important for reemergence from latency and
persistent infection in vivo by -herpesviruses. The acute, latent,
reemergent, and chronic phases of infection of the murine herpesvirus
HV68, which encodes a D-type vCYCLIN and a vBCL-2 (Virgin et al. 1997),
have been examined in vivo. HV68 viral stop-codon and frame-shift
mutants that lack either the v-cyclin or v-bcl-2
display no prominent defects in acute replication in vitro or in vivo
in wild-type or immunocompromised mice (Hoge et al. 2000; Van Dyk et
al. 2000; Gangappa et al. 2002). EBV BHRF1 is also dispensable for
acute virus replication (Marchini et al. 1991; Lee and Yates 1992).
Establishment of HV68 viral latency was also unaffected by
v-cyclin or v-bcl-2 mutations (Hoge et al. 2000; Van
Dyk et al. 2000; Gangappa et al. 2002). It is probably not surprising
that vBCL-2 function is not required during acute replication and
establishment of latency, given that herpesviruses encode multiple gene
products that are probably functionally redundant inhibitors of
apoptosis, including LMP1 and vFLIP. Alternatively, modulation of the
cell cycle and apoptosis by the virus may be unnecessary for these
aspects of the virus life cycle.
In contrast to their dispensability in acute and latent infection, both
the HHV8 v-cyclin and v-bcl-2 are required for
efficient reemergence from latency, persistent infection, and
pathogenesis. HV68 v-cyclin or v-bcl-2 mutant viruses
display significant defects in reemergence from latency as measured by
spontaneous ex vivo reactivation, and in persistent replication and
virulence (Hoge et al. 2000; Van Dyk et al. 2000; Gangappa et al.
2002). Because latently infected cells are not cycling, it is possible
that v-cyclin expression may be required to stimulate cell
cycle progression; however, it may also induce apoptosis, thereby
necessitating the presence of an antiapoptotic vBCL-2. Indeed, HV68
or HHV8 vCYCLIN overexpression stimulates cellular DNA synthesis and
apoptosis (Ojala et al. 1999; Van Dyk et al. 1999), as does adenovirus
E1A (White 1994, 2001). Direct assessment of apoptosis induction by vCYCLIN expression in a v-bcl-2 mutant but not in wild-type
infected cells during reemergence from latency would be informative,
but may be technically challenging. Furthermore, if apoptotic signaling by vCYCLIN requires BAX and/or BAK, and there are indications that
herpesvirus vBCL-2s may interact with and inhibit BAX and BAK, it will
be of interest to evaluate the herpesvirus life cycle in cells or
animals deficient for BAX and/or BAK expression. A prediction is that
BAX and BAK deficiency should rescue defective reemergence from
latency, persistent infection, and pathogenesis of a v-bcl-2
mutant herpesvirus. Given that these are aspects of the herpesvirus
life cycle that are associated with human disease, they present an
opportunity for therapeutic intervention.
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Inhibition of death receptor signaling by vBCL-2 proteins |
Immune surveillance by the host organism would partially entail
attacking the infected cell with secreted TNF-, FasL, or TRAIL. One
possible function of the vBCL-2 proteins, therefore, would be to
prevent death of the infected cell due to signaling by death cytokines.
There are several examples of inhibition of death receptor signaling by
vBCL-2 proteins. The BHRF1 protein of EBV is capable of inhibiting both
TNF-- and FasL-induced death, but its activity may be
cell-type-specific (Foghsgaard and Jäättelä 1997; Kawanishi
1997). BALF1, also encoded by EBV, is capable of blocking FAS-mediated
death signaling in cells sensitized by IFN (Marshall et al. 1999).
The M11 protein of HV68 can inhibit TNF- and FasL-induced death
(Wang et al. 1999), and the vBCL-2 of HVS blocks FAS signaling in a
cell-type-dependent manner (Derfuss et al. 1998). The presence of
redundant antiapoptotic functions in many viral genomes makes it
unclear what the overall contribution of vBCl-2 proteins is to
inhibition of cytokine death signaling during infection. It remains
possible that inhibition of apoptosis mediated by death receptors by
vBCL-2s is an indirect consequence of inhibition of the core apoptotic
machinery represented by BAX and BAK, which may block yet another
apoptotic pathway. Alternatively, it may be so critically important to
block apoptosis by death receptor ligands that viruses encode redundant
inhibitory mechanisms to ensure survival of the infected cell.
The system in which inhibition of death receptor-mediated apoptosis by
vBCL-2s has been explored most thoroughly is with adenoviruses. In
human cells, expression of the adenovirus type 2 or 5 E1B 19K potently
inhibits apoptosis induced by TNF-, anti-FAS antibodies, and TRAIL
(Gooding et al. 1991; Hashimoto et al. 1991; White et al. 1992;
Tollefson et al. 2001
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Introduction
Identification of vBCL-2s
