Regulation of papillomavirus
transcription and replication; insights for the design of extrachromosomal
vectors
Review
Article
Alison A.
McBride
__________________________________________________________________________________
Received: 16 October 1998;
accepted: 20 October 1998
The papillomaviruses infect
and replicate in the stratified layers of skin and mucosa and give rise to benign
lesions called warts or papillomas. The virus infects basal epithelial cells
and within these persistently infected cells the viral genome is maintained at
low levels as extrachromosomally replicating viral DNA. The genomes of
papillomaviruses can also be stably maintained as high copy number
extrachromosomal elements in cell lines and within these cells the viral
genomes replicate in synchrony with cellular DNA. The E1 and E2 viral proteins
regulate viral transcription, initiation of replication and long term episomal
maintenance of viral genomes. This review will describe the functions of the E1
and E2 proteins and discuss how these functions can be exploited in the design
of extrachromosomal replicating vectors for gene therapy.
Certain DNA viruses, such as papillomavirus or
Epstein-Barr virus, are able to maintain their genomes as stable
extrachromosomal elements in the nuclei of infected cells. The papillomaviruses
are small DNA viruses that infect basal epithelial cells and replicate in
terminally differentiating keratinocytes. These viruses have been isolated from
a wide range of vertebrates and they exhibit both host species and tissue
specificity. Viral DNA replication has been studied mostly in bovine
papillomavirus type 1 (BPV-1) and the human papillomaviruses (HPV), HPV-1, -11,
-16 and -18 and -31.
The viral E1 and E2 proteins are important for
initiation of viral DNA replication and for regulation of viral transcription.
The E1 protein is the primary viral replication initiator protein (Ustav and
Stenlund, 1991a; Mohr et al., 1990;
Yang et al., 1991) and E1 also
functions as a transcriptional repressor (Sandler et al., 1993; Le Moal et al.,
1994); the viral E2 protein(s) are transcriptional regulatory proteins that
regulate the expression of the other viral gene products and, in addition, play
an important role in DNA replication. The E2 transactivator protein is also
required for long-term episomal maintenance of viral genomes within replicating
cells (Piirsoo et al., 1996).
Papillomavirus genomes and the E2-TA protein interact with mitotic chromosomes
in dividing cells and this association is likely to be important for genome
segregation (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998).
Papillomaviruses infect and replicate in stratified
epithelium and give rise to benign lesions called warts or papillomas.
Papillomaviruses infect the lower basal layer of cells of a stratified
epithelium (Figure 1). The a6b4
integrin protein, expressed exclusively in this cell layer, acts as a receptor
for the virus (Evander et al., 1997).
Damage to the superficial layers of the epithelium is probably necessary to
allow access of virus to the basal layer. Within basal cells the viral genome
is amplified to a low copy number and maintained as an extrachromosomally
replicating circle of double stranded DNA (Figure
2). DNA replication in these cells probably requires the viral E1 and E2
replication proteins. The viral E5 protein is also expressed in basal cells. E5
stimulates the activity of growth factor receptors
expressed by the cell and induces cellular proliferation (reviewed in Howley,
1995). Enhanced proliferation of basal cells may be important to increase the
population of infected cells and to provide a suitable environment for
establishment of a productive viral lesion. As basal cells differentiate and
migrate up to the stratum spinosum, expression of the E2 proteins is greatly
increased and vegetative DNA replication begins (Burnett et al., 1990; Howley, 1995). The cells in this layer do not
normally divide nor express cellular proteins necessary for DNA replication.
Therefore, the viral E7 protein is required to induce the differentiated
keratinocytes to enter S-phase and synthesize cellular replication proteins by
binding to and inactivating the cellular retinoblastoma protein, Rb (reviewed
in Jones and Munger, 1996). However, the conflicting signals of cell cycle
progression and differentiation induce the p53 protein, which in turn signals
cells to undergo apoptosis or growth arrest. The viral E6 protein can
inactivate this function of p53 by targeting it for degradation by the
ubiquitin-proteasome pathway (reviewed in Kubbutat and Vousden, 1996). The
viral E4 protein is also abundant in the more differentiated layers of a
papilloma. It has been hypothesized that E4 may function as a nuclear
structural protein, an RNA splicing and transport factor, or in release of
viral particles from the papilloma (reviewed in Howley, 1995). In the upper
differentiated layers of the papilloma, the viral capsid proteins L1 and L2 are
synthesized and virions are assembled (reviewed in Howley, 1995).
Three modes of DNA replication take place in the
papillomavirus life cycle: initial DNA amplification, maintenance replication
and vegetative replication. After initial uptake of the virus, the virion
particle is uncoated and the genome transported to the nucleus of the basal
cell where it is presumed to be amplified to a low copy number (Zhou et al., 1995). Presumably, a low level
of the E1 and E2 proteins must be expressed early after infection since there
is no evidence that they are in the viral particle. Most experimental studies
have examined transient DNA replication in cultured cells, a system that is
probably most analogous to this initial amplification stage and which requires
the E1 and E2 proteins and the viral replication origin (Ustav and Stenlund, 1991a;
Ustav and Stenlund, 1991a).
Stable episomal maintenance is the second stage of
papillomavirus DNA replication. In a papilloma, the infected basal cells
proliferate and maintain low levels of extrachromosomal viral DNA. The genomes
of papillomaviruses can also be stably maintained as high copy number
extrachromosomal elements in cell lines (Law
et al., 1981) and within these lines the viral genomes replicate in
synchrony with cellular DNA. The viral genome copy number remains constant
overall but the genomes are replicated by a random choice mechanism (Gilbert
and Cohen, 1987; Ravnan et al.,
1992). Long term, stable maintenance of papillomavirus-derived plasmids
requires expression of the E1 and E2 proteins, the replication origin and a
region from the LCR, that has been designated a minichromosome maintenance
element (MME) (Piirsoo et al., 1996).
This element contains multiple high affinity E2 binding sites. Recent studies
have shown that both the BPV1 E2 transactivator protein and BPV genomes are
associated with cellular chromosomes at mitosis (Skiadopoulos and McBride,
1998; Lehman and Botchan, 1998). This could be the mechanism by which
approximately equal numbers of viral genomes are segregated to daughter cells
at cell division to ensure that all basal cells of a papilloma contain viral
DNA .
The third stage of viral replication is vegetative DNA
replication, which is required to generate progeny virus. Vegetative DNA
replication only occurs as the basal cells of a papilloma migrate upwards and
differentiate in the stratum spinosum layer. Increased expression of the E2
proteins also occurs in the stratum spinosum and may be important for
amplification of viral DNA (Burnett et
al., 1990). The E2 protein is important for initiation of viral DNA replication
but it has also been shown that HPV-31 E2 can arrest cells in S phase (Frattini et al., 1997). Clearly this could be
important for vegetative replication by allowing sustained synthesis of viral
DNA. There appears to be a switch from bidirectional theta replication in the
maintenance stage of replication to a rolling circle mode in the vegetative
stage (Flores and Lambert, 1997). Little else is known about vegetative viral
DNA replication because of the requirement for terminally differentiating keratinocytes
and difficulties in reproducing these conditions in a culture system. However,
great advances are being made by replicating papillomaviruses in organotypic
raft cultures and in xenografts of mice and these systems are proving to be
very useful in studying the entire viral life cycle (reviewed in Meyers and
Laimins, 1994).
Papillomavirus transcription is regulated primarily by
the viral E2 gene products. These proteins regulate transcription by binding to
specific DNA sites located in the viral genomes (see Figure 2). In bovine papillomavirus type 1 several gene products
are expressed from the E2 ORF and they have been shown to function as
transcriptional activators and repressors (Figure
3). cDNA species that could potentially encode truncated human
papillomavirus E2 repressor proteins have been cloned but, as yet, no such
proteins have been identified. Some HPVs may have evolved a mechanism to both
activate and repress viral transcription with the full-length E2 protein (see Figure 5, reviewed in McBride and
Myers, 1997; Fuchs and Pfister, 1994).
The full-length E2 protein from all papillomaviruses
consists of a 200 amino acid N-terminal transactivation domain linked to a 100
amino acid C-terminal DNA binding and dimerization domain by a flexible hinge
region of variable length and sequence (reviewed in McBride and Myers, 1997;
McBride et al., 1989; Dostatni et al., 1988). The E2-TA protein
activates transcription by binding to specific DNA binding sites that are
located within enhancer elements in the viral genome (reviewed in McBride and
Myers, 1997). There are seventeen different E2 binding sites in the BPV-1
genome that vary in affinity for the E2 protein over two orders of magnitude
(Li et al., 1989) (Figure 2). The well-studied genital-associated HPV genomes contain only
four E2 sites in the LCR (Figure 5).
The C-terminal domain of E2 binds specifically to DNA
as a dimer. The X-ray crystal structure of the C-terminal 85 amino acids of E2
bound to DNA was the first example of an anti-parallel b-barrel DNA binding structure (Hegde et al., 1992). The DNA binding domain
forms an eight-stranded anti-parallel b-barrel made up of four strands from each subunit. A pair of a-helices symmetrically positioned on the outside of
the barrel contain the amino acids residues that are required for specific DNA
interaction. The DNA binding domain of the Epstein Barr virus EBNA1 protein has
a very similar structure to the E2 DNA binding domain despite no sequence
similarity (Bochkarev et al., 1995).
The 200 amino acid E2 transactivation domain, unlike
many other transactivation domains, appears to have a very constrained
structure that is easily disrupted by deletion or certain non-conservative
point mutations (reviewed in McBride and Myers, 1997). The transactivation
domain is also critical for the replication function of the E2 protein and for
interaction with the E1 protein (reviewed in McBride and Myers, 1997). The
exact mechanism of transactivation has not been elucidated but probably
involves interaction with components of the basic transcriptional machinery.
BPV1 E2 has been shown to interact with SP1, TBP, TFIIB and a novel cellular
protein, AMF-1 (Li et al., 1991;
Steger et al., 1995; Rank and
Lambert, 1995; Breiding et al.,
1997).
In BPV-1, the E2 ORF encodes three different polypeptides;
the E2-TA transactivator protein is encoded by the entire ORF and two smaller
polypeptides, E2-TR and E8/E2, are encoded by the 3' half of the ORF. The
shorter polypetides function as transcriptional repressors by antagonizing the
function of E2-TA (Hubbert et al.,
1988; Lambert et al., 1987; Spalholz et al., 1985; Lambert et al., 1989; Choe et al., 1989). The repressors contain only the DNA
binding/dimerization domain and function both by direct competition with E2-TA
for binding to the E2 DNA binding sites and by the formation of inactive
heterodimers with the full-length E2-TA protein (Lim et al., 1998; Barsoum et al.,
1992) (Figure 5).
In several HPVs associated with the anogenital tract,
the full length E2 protein appears to repress the promoter located upstream
from the E6 gene (reviewed in McBride and Myers, 1996, 1997; Fuchs and Pfister,
1994). This probably occurs when the E2 dimer binds to E2 DNA binding sites
that overlap binding sites for the cellular SP1 and TFIID transcription
factors. Recent studies have indicated that these proximal E2 binding sites
have lower affinity for the E2 protein than the E2 binding sites are located
further upstream from the promoter start site. This has led to a model in which
low levels of E2 bind to the higher affinity upstream E2 sites and activate
transcription, but at high levels of E2 protein the lower affinity proximal E2
sites are occupied leading to transcriptional repression (Figure 5). The situation
is probably even more complex in a papilloma as the levels and activities of
the E2 proteins and cellular transcription factors are likely modulated by cell
cycle and epithelial differentiation.
Figure 5. Mechanisms of transcriptional regulation by the papillomavirus E2
proteins. A. BPV1 expresses a transcriptional
transactivator with a transactivation domain and DNA binding/dimerization
domain. Two shorter repressor proteins contain only the DNA
binding/dimerization domain and repress E2 transactivation by forming
heterodimers with the transactivator and by competing for the binding to the E2
sites in the viral genome. B. In
many HPVs the full-length E2 protein can activate transcription by interacting
with higher affinity E2 binding sites upstream from the transcriptional start
site. At higher levels of E2, the lower affinity sites close to the promoter
become occupied. This displaces essential cellular factors, SP1 and TFIID and
results in repression of basal promoter activity.
Figure 6. Model of initiation of viral DNA replication. The E1 and E2 proteins
initiate DNA replication by cooperatively binding to specific sites in the
viral origin of replication. It has been proposed that an E1-alone complex then
assembles in a ring-like hexamer structure around the DNA and the helicase
activity of E1 unwinds the origin to allow access of the cellular replication
machinery.
The viral E1 replication protein can also function as
a transcriptional repressor. Inactivation of E1 increases the immortalizing or
growth transforming potential of HPV-16 and BPV-1, respectively (Schiller et al., 1989; Lambert and Howley, 1988;
Romanczuk and Howley, 1992) and this correlates well with the frequent
disruption of E1 and/or E2 expression found in HPV-associated carcinomas. The
E1 protein of BPV-1 can repress E2-mediated transactivation of the viral P89
promoter, which expresses the E6 and E7 gene products (Sandler et al., 1993; Le Moal et al., 1994). This is probably a
consequence of binding of an E1/E2 complex to the replication origin, which is
located just upstream from P89.
In addition to the cellŐs replication machinery,
papillomavirus DNA replication requires the full-length E2 transactivator
protein, the viral E1 protein and the replication origin (Ustav and Stenlund,
1991a; Ustav et al., 1991b; Ustav and
Stenlund, 1991a). The minimal origin of replication consists of an E1 binding
site, an E2 binding site and an AT rich region that may facilitate origin
unwinding. (Ustav et al., 1991b). The
E1 protein has several replication-associated activities such as
origin-specific binding (Wilson and Ludes-Meyers, 1991) and helicase activities
(Yang et al., 1993) and forms a
complex with the E2 transactivator (Mohr
et al., 1990; Blitz and Laimins, 1991; Seo et al., 1993; Spalholz et
al., 1993; Sedman and Stenlund, 1995) (Figure
4). The E1 and E2 sites have relatively low affinity for their respective
proteins but together they cooperatively bind to the origin with high affinity
(Figure 6). After the initial
binding of an E1/E2 complex to the origin, the E1 protein oligomerizes to form
a trimer or hexamer that encircles the DNA and E2 dissociates from the origin
(Sedman and Stenlund, 1996, 1998). The E1 helicase function of the hexamer then
unwinds the DNA at the origin to allow DNA synthesis to begin (Sedman and
Stenlund, 1998) (Figure 6).
The E2-TA transactivator plays an auxiliary role in
replication by enhancing and regulating the functions of the E1 protein. In
addition to cooperatively binding to the origin with the E1 protein, E2
alleviates repression of replication by nucleosomes (Li and Botchan, 1994) and
interacts with cellular replication proteins such as RPA (Li and Botchan,
1993).
Rodent cells transformed by BPV-1 maintain
approximately 50 to 200 copies of the viral genome indefinitely as
extrachromosomal nuclear plasmids (Law et
al., 1981). Cell lines derived from cervical carcinomas can also maintain
human papillomavirus genomes as extrachromosomal elements (Bedell et al., 1991). Plasmids containing the
minimal viral replication origin replicate transiently in cells expressing the
E1 and E2 proteins but the replicated DNA is lost with time. Long-term stable
maintenance of origin-containing plasmids also requires regions from the LCR
that contain multiple high affinity E2 DNA binding sites in addition to the
replication origin (Piirsoo et al.,
1996). This region has been designated the minichromosome maintenance element
(MME) and can be substituted by ten tandem copies of E2 DNA binding sites,
suggesting that the E2 protein and the E2 DNA binding sites are important for
genome segregation (Figure 7). This
finding is supported by the observation that the E2-TA protein and BPV-1
genomes are associated with
condensed mitotic chromosomes in dividing cells
(Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998) (Figure 8) and supports a model in which
viral genomes are attached to mitotic chromatin indirectly via the E2 protein
and E2 DNA binding sites (Figure 9).
This interaction would ensure that approximately equal numbers of viral genomes
are segregated to daughter cells. Viral genomes that replicate as
extrachromosomal plasmids may also require a mechanism to ensure that they are
not lost from the nucleus during cell division. Association with cellular
chromosomes would ensure that viral genomes are enclosed in the nuclear
membrane during telophase. The genomes may also interact with some cellular
component that ensures that they are in a transcriptionally active region of
the nucleus as the cells move into the G1 stage of the cell cycle.
The BPV-1 E2-TA protein interacts with mitotic
chromatin in the absence of viral genomes. Conversely, the E2-TR and E8/E2
proteins are dispersed throughout the cell during mitosis and are excluded from
mitotic chromatin. This indicates that the DNA binding domain of the E2 protein
is not sufficient for the interaction with mitotic chromosomes and suggests
that the interaction is not mediated by binding to cellular DNA sequences. The
finding that a DNA-binding defective E2-TA protein retains the ability to
interact with mitotic chromatin also supports this. Furthermore, deletions
within the N-terminal domain abrogate the ability of E2 to interact with
mitotic chromosomes. These findings indicate that the N-terminal
transactivation domain of E2-TA is necessary for the interaction (Skiadopoulos
and McBride, 1998).
As yet, it is not known what component of mitotic
chromatin is important for interaction of the E2 protein with mitotic
chromatin. One possibility is that E2 interacts with some constituent of the
chromosomal scaffold or chromosomal periphery. The chromosomal periphery is a
region around the condensed chromatids that contains many proteins, some of
which form a network of fibrils and granules (Hernandez-Verdun and Gautier,
1994). Several components of the nuclear matrix are found in the
perichromosomal region as well as a number of ŇpassengerÓ proteins from the
nucleus and nucleoli. The E2-TA protein (but not the E2-TR or E8/E2 proteins)
has been shown to be associated with the nuclear matrix (Hubbert et al., 1988) and it will be interesting
to determine whether the same interactions are important for the association
with mitotic chromosomes. Nuclear matrix attachment sites have also been
identified in the BPV-1 genome (Adom and Richard-Foy, 1991; Adom et al., 1992; Tan et al., 1998) and it is possible that these sites are also
important for interaction of the genomes with mitotic chromatin instead of, or
in addition to, E2 DNA binding sites.
Although the overall viral copy number in a population
of BPV-1 transformed cells remains relatively constant, several studies have
shown that individual cells contain a wide range of copy numbers (Roberts and
Weintraub, 1988; Ravnan et al., 1992;
Ravnan and Cohen, 1997). Stewart et al.
(1994) also demonstrated that there is significant randomization in replication
and/or partitioning. This suggests that segregation does not occur by a very
precise mechanism and is consistent with the model that the E2 proteins and
viral genomes randomly associate with mitotic chromatin as passenger molecules.
This model would also predict that the viral copy number depends on the levels
of the E2-TA protein.
A similar phenomenon has been observed for
Epstein-Barr virus (EBV). EBV infects and immortalizes B-lymphocytes and the
viral genome is maintained indefinitely as an extrachromosomal element. The
EBNA-1
Figure 8. Papillomavirus genomes and the E2-TA transactivator protein are
associated with cellular chromosomes in mitotic cells. E2 proteins were
detected in COS7 cells expressing the E2-TA protein by indirect
immunofluorescence using an E2-specific antibody. Panels A and B show COS-7 cells
as a control. Panels C and D show COS-7 cells expressing E2-TA. In
panels A and C, cellular DNA was detected by propidium iodide staining. In
panels B and D, FITC-labeled E2 protein is detected in the same field of cells.
BPV DNA was detected by fluorescent in situ hybridization in C127 cells (E and F) and 137 cells (that contain BPV-1) (G and H). In panels E
and G cellular DNA was detected by the propidium iodide signal. In panels F and
H the same field of cells are shown with the FITC-labeled BPV DNA signal.
Mitotic cells are indicated by white arrows.
Figure 9. This diagram shows a model in which papillomavirus genomes are
linked via the E2-TA protein to condensed mitotic chromosomes.
protein of EBV is a transcriptional transactivator and
a replication protein and it is the only viral protein required for replication
and maintenance of plasmids containing the oriP origin of replication (which
contains a number of repeated EBNA DNA binding sites) (Yates et al., 1985). The EBNA 1 protein and
EBV genomes have also been shown to be randomly associated with mitotic
chromatin (Grogan et al., 1983;
Harris et al., 1985) and it has been
suggested that these properties might be important for the genome segregation
and nuclear retention function of EBNA-1. The EBNA-1 protein also promotes
prolonged nuclear retention of plasmids containing EBNA-1 DNA binding sites but
no origin of replication (Krysan et al.,
1989; Middleton and Sugden, 1994) and it has been proposed that this is due to
the interaction of plasmids with mitotic chromosomes.
The Epstein-Barr virus EBNA-1 protein and the
papillomavirus E2-TA protein have common roles in the life cycles of their
respective viruses (Grossman and Laimins, 1996). Both proteins are
transcriptional transactivators that activate transcription by binding to specific
binding sites within the viral genomes. Notably, both proteins have dimeric DNA
binding. domains with almost identical anti-parallel b-barrel structures, despite no amino acid homology
(Bochkarev et al., 1995). Both
viruses replicate and maintain their genomes as extrachromosomal elements in
persistently infected cells. This maintenance requires both E2-TA and the
multiple E2 binding sites in the MME element of papillomavirus (Piirsoo et al., 1996) or EBNA-1 and the
multiple EBNA-1 binding sites in the oriP element of Epstein-Barr virus (Yates et al., 1985). In both cases the viral
proteins and genomes are associated with condensed cellular chromosomes during
mitosis (Skiadopoulos and McBride, 1998; Lehman and Botchan, 1998; Grogan et al., 1983, Harris et al., 1985). The EBNA-1 protein
promotes prolonged nuclear retention of plasmids containing EBNA-1 DNA binding
sites even in the absence of replication (Krysan et al., 1989; Middleton and Sugden, 1994) and it has been proposed
that this is due to the association with mitotic chromosomes.
Gene therapy vectors that replicate and
are retained extrachromosomally have several advantages over those that
integrate in a random fashion into the host genome. These vectors will persist
in proliferating cells and should not generate mutations by insertion into the
cellular chromosomes. Such vectors can be maintained at a high copy number and
are not susceptible to positional effects, such as inactivation, that are dependent
on the integration site. EBV-based vectors that contain the EBNA-1 gene and the
oriP replication origin have been developed and can express foreign gene
products in primate and human cells (reviewed in Calos, 1996). Another class of
EBV vectors have been developed that only contain the EBNA-1 gene and repeats
of the EBNA-1 binding site required for nuclear retention. In these vectors the
oriP origin has been replaced with a cellular replication origin and the
resulting vectors are able to replicate in a wider range of mammalian cells
(Krysan et al., 1989). The EBNA-1
protein also has the advantage that it is not recognized by the cell-mediated
immune system (Levitskaya et al.,
1995) as it is resistant to the proteasome-mediated degradation that is required
for antigen presentation (Levitskaya et
al., 1997). However, there is a report that the EBNA-1 protein can cause
lymphomas in transgenic mice expressing this protein in B-cells (Wilson et al., 1996).
Sarver et al. (1981) first described the use of papillomaviruses as
vectors in 1981. In general, these vectors comprised the 69% transforming
region of the virus (the genome minus the late region) and the foreign gene to
be expressed. A newer vector only contains the LCR and the E1 and E2 genes (Ohe et al., 1995). However, these vectors
have a limited host range and, in some cases, insertion of a foreign
transcriptionally active foreign gene causes the plasmid to integrate
(Waldenstrom et al., 1992). This is
probably because the small papillomavirus genomes are very compact and contain
multiple overlapping genes and regulatory signals that can be inadvertently
disrupted. The presence of an active heterologous enhancer and promoter could
interfere with viral replication by transcriptional interference. Using the
detailed knowledge of the mechanisms of papillomavirus replication and genome
maintenance, it should be possible to generate a new class of papillomavirus
vectors. These vectors could express the E1 and/or E2 genes from different
promoters suitable for a specific cell type and either viral or cellular
replication origins could be incorporated, as has been described for EBV-based
vectors (Krysan et al., 1989). The
addition of repeated E2 binding sites may be sufficient to maintain the vector
as an episome when either a viral or cellular replication origin is used.
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