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Journal of Virology, October 2001, p. 9446-9457, Vol. 75, No. 19
Medical Scientist Training Program, Cell and
Molecular Biology Graduate Program, Department of Microbiology and
Immunology, and Comprehensive Cancer and Geriatrics Center, University
of Michigan Medical Center, Ann Arbor, Michigan 48109-0934
Received 9 May 2001/Accepted 5 July 2001
The latency-associated nuclear antigen (LANA) encoded by the
Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the
majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for
B-lymphocyte transformation, is expressed. EBNA2 upregulates the
expression of LMP1 and other cellular genes through
specific interactions with cellular transcription factors tethering
EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we
demonstrated that LANA can efficiently transactivate both the LMP1 and
C promoters in the human B-cell line BJAB as well as in the
human embryonic kidney 293 cell line. Moreover, we demonstrated that
specific domains of LANA containing the putative leucine zipper and the
glutamic acid-rich region are highly effective in upregulating
these viral promoters, while the amino-terminal region (435 amino
acids) exhibited little or no transactivation activity in our assays.
We also specifically tested truncations of the LMP1 promoter element
and showed that the Epstein-Barr virus (EBV) and
Kaposi's sarcoma-associated herpesvirus (KSHV; also referred to as
human herpesvirus type 8) are two closely related human
gammaherpesviruses (10, 49, 61). EBV primarily infects
nasopharyngeal epithelia and B lymphocytes and is associated with a
number of human malignancies, including nasopharyngeal carcinoma,
Hodgkin's disease, Burkitt's lymphoma, immunoblastic lymphomas,
T-cell lymphomas, gastric carcinoma, breast carcinoma, and body
cavity-based lymphomas (BCBLs) (3, 9, 37, 56, 82, 84). EBV
typically establishes distinct types of viral latency based on specific
latent-gene expression profiles in the infected cells
(56). These latent-gene expression profiles include the
expression of EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C, and LP,
the EBV early RNAs (EBERs), latent membrane proteins (LMPs) 1, 2A, and
2B, and BARF0 transcripts (56). LMP1 is essential for
mediating EBV-induced proliferation of infected B cells through
ligand-independent events and signal transduction (37,
48). EBNA1 is required for episomal maintenance and stability of
the newly replicated episomes, ensuring that they are not lost during
segregation of progeny cells (1, 26, 32, 39, 83). EBNAs
LP, 2, 3A, and 3C are critical whereas the EBERs, EBNA-3B, LMP2A,
LMP2B, and BARF0 are dispensable for growth transformation of primary B
cells (12, 22, 37, 43-45, 59, 60, 70, 72).
Typically, in healthy individuals, the virus remains tightly latent and
proliferation of the infected B cell is controlled by the cytotoxic
T-cell response (36, 56). However, in the case of an
immunocompromised patient, this stringently balanced mechanism is
disrupted and the infected cells are dysregulated, leading to
proliferation in the infected host (11, 56). Previous studies have shown that BCBLs establish a predominantly type II latency
program (5, 24), although one report indicated that a type
I latency program was established in one BCBL cell line (71). Type II latency is characterized by the expression
of EBNA1, the LMPs, and BARF0 transcripts and is notably lacking expression of EBNA2 (37). This type of infection is seen
in all non-B cells infected with EBV, such as in T-cell lymphomas, Hodgkin's disease, and nasopharyngeal as well as other carcinomas (56). The expression of the entire repertoire of latent
antigens is considered to be a type III latency program and is
typically seen in lymphoblastoid cell lines (LCLs) and in
lymphoproliferative disorders occurring in AIDS patients and others
with severe immunosuppression (56). Expression of the
EBERs and BARF0 transcripts is usually seen in the majority of
EBV-associated cancers, with few exceptions (11, 56).
LMP1 of EBV is an integral membrane protein that is essential for
growth transformation of B lymphocytes (27, 34, 78). This
viral oncogene is involved in triggering many of the morphologic changes associated with EBV infection, including the expression of
adhesion molecules and activation markers as well as NF- Previous genetic and biochemical studies have demonstrated that EBNA2
is essential for EBV-mediated growth transformation of B lymphocytes
and is a potent activator of transcription in EBV infection (12,
22). EBNA2 lacks the ability to directly bind DNA but targets
and activates EBV promoter elements through its association with
RBP-J The two major essential latent EBV promoters, the C promoter and the
LMP1 promoter, generate the transcripts of the EBNA family and LMP1
during latent infection in vitro and are also active in lesions of
patients with lymphoproliferative disease (21, 23, 37, 56, 81,
85, 89). Both of these EBV latent promoters are upregulated
through indirect interaction with EBNA2 (29, 76). The
interaction between EBNA2 and the LMP1 promoter is dependent on the
PU.1 Ets family protein, the RBP-J KSHV is the potential etiological agent of Kaposi's sarcoma and is
associated with primary-effusion lymphomas (PELs; also referred to as
BCBLs) and multicentric Castleman's disease; more controversially, it
has been found in dendritic cells of individuals with multiple myeloma
(7, 10, 17, 19, 55, 67). The KSHV genome is detected in
endothelial and tumor cells of Kaposi's sarcoma lesions, where the
virus expresses a number of critical viral antigens during a persistent
latent infection (9, 24, 54, 68, 88). Similar to EBV, KSHV
shows distinct patterns of gene expression in different KSHV-associated
diseases (33, 50). However, the latency-associated nuclear
antigen (LANA) is constitutively expressed in all known KSHV-infected cells.
LANA is encoded by KSHV open reading frame 73 and is required for the
persistence of the KSHV episome in latently infected cells (35,
52). LANA is a large nuclear phosphoprotein with three distinct
domains (Fig. 1): a proline-rich
N-terminal region that contains a putative nuclear localization signal
(NLS), a repeating internal acidic domain that is rich in glutamic
acid, and a carboxy terminus containing a potential leucine zipper
motif (61). Both the N and C termini are sufficient to
localize LANA to the nucleus of the host cell (63). The
efficient segregation of KSHV episomes to progeny cells during mitosis
is most likely facilitated by LANA via tethering of KSHV DNA to host
chromosomes (2, 13). It was also recently shown that LANA
inhibits p53 function, disrupting the cell death pathway and thereby
contributing to the persistence of KSHV (18) and
associated oncogenes. Additionally, LANA can regulate an E2F-responsive
promoter through association with the retinoblastoma protein,
suggestive of functions similar to the large T antigen of the
polyomaviruses (51).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.19.9446-9457.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Latency-Associated Nuclear Antigen Encoded by
Kaposi's Sarcoma-Associated Herpesvirus Activates Two Major
Essential Epstein-Barr Virus Latent Promoters
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
204 to +40 region had increased levels of
activation compared with a larger region, 512 to +40, which contains
two recombination signal-binding protein J
binding sites. The
smaller, 204 to +40 promoter region contains specific binding sites
for the Ets family transcription factor PU.1, transcription
activating factor/cyclic AMP response element, and Sp1, all of which
are known to function as activators of transcription. Our data
therefore suggest a potential role for LANA in regulation of the major
EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore,
LANA may be able to activate transcription of viral and cellular
promoters in the absence of EBNA2, potentially through association with
transcription factors bound to their cognate sequences within the 204
to +40 region. This regulation of viral gene expression is critical for
persistence of these DNA tumor viruses and most likely involved in
mediating the oncogenic process in these coinfected cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B activation
(16, 28, 78, 79). The transforming ability of LMP1 is
tightly linked to its association with the tumor necrosis factor family
receptor-associated factors and may function similarly to the tumor
necrosis factor family receptor, aggregating in the plasma membrane,
where it is capable of binding tumor necrosis factor family
receptor-associated factors (15, 48). Numerous studies of
LMP1 regulation have demonstrated that expression of LMP1 is dependent
on regulation of its promoters by viral and cellular transcription
factors (20, 21, 30, 42, 64, 65, 76).
and other cellular transcription factors, including PU.1 and
AML1 (21, 23, 30, 42, 64-66, 81). EBNA2 also
associates with the basal transcription machinery and other
coactivators of transcription and has been shown to transactivate the
major EBV latent promoters and other responsive cellular promoters (73-75).
transcription factor, and other
factors which target their cognate sequences within the promoter
regulatory region (23, 30, 64). Transactivation is
significantly reduced in the absence of the RBP-J consensus sites,
and the transcription activating factor/cyclic AMP response element
(ATF/CRE) has also been shown to mediate EBNA2-dependent and
-independent activation of LMP1p (30, 66).
View larger version (22K):
[in a new window]
FIG. 1.
Schematic diagram of LANA showing its structural
properties. Note that the clone consisting of amino acids 1 to 435 contains the proline-rich region (P-rich), a nuclear localization
sequence (NLS), and part of the acidic region (AD). The LANA
construct consisting of amino acids 301 to 942 contains the entire
acidic domain, including the glutamine-rich region (Q-rich) and the
entire putative leucine zipper (L-zip). The amino acid 762 to 1162 clone contains the putative leucine zipper and a potential nuclear
localization sequence. The LANA construct with amino acids 1 to 756 lacks the putative leucine zipper but contains the acidic domains, the
proline-rich region, and a nuclear localization sequence. The amino
acid 1 to 950 clone is identical to the amino acid 1 to 756 construct
except that it includes the putative leucine zipper.
Coinfection of KSHV and EBV has been seen in the majority of AIDS-associated PELs and in a minority of cases not associated with AIDS (6, 19, 24, 62). This unique dual herpesvirus infection provides a useful model to study the interaction between these two human DNA tumor viruses. In the case of KSHV- and EBV-coinfected cells, many of the major essential EBV latent antigens are not detected (5, 24, 71). However, EBNA1 and LMP1 expression was shown to occur, but at significantly lower levels than in cells infected with only EBV (5). In the absence of viral proteins EBNA2, EBNA3A, and EBNA3C, it is likely that KSHV plays a very significant role in regulating EBV gene expression, hence contributing to the pathogenesis of PELs. This body of data suggests that KSHV-encoded proteins or KSHV-induced cellular proteins might regulate EBV latent gene expression. Due to the ubiquitous expression of LANA in KSHV- and EBV-coinfected cells, we chose to investigate this protein in terms of its role in regulating gene expression from two major EBV latent promoters.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Plasmids.
The pGL reporter plasmids (Promega Inc.) contain
the luciferase gene and lack eukaryotic promoter and enhancer
regulatory sequences. The LMP1 and C promoter elements, which were
cloned into the basic reporter plasmid to assess the ability of LANA to
influence expression from these promoters by using luciferase assays,
were described previously (41, 80). The pGL2 512/+72 plasmid has two directly repeated copies of the LMP1 512/+72 promoter
element, and pGL 512/+40 contains a single copy of the 512/+40
promoter element (obtained from Jeffery Lin and Elliott Kieff). pGL
272/+40 and pGL 204/+40 have truncations of the 512/+40 LMP1
promoter region, (consecutive deletions of the two RBP-J binding
sites) (30). The reverse primer
5'-TATAGATCTCTCAGGGCAGTGTGTCAGGAG-3' was used along with the
forward primer 5'-TAGGTACCCTGATTGCCGCACTGCCTTTCC-3' or
5'-TAGGTACCGGGACCCGCTTTTCTAACACAAAC-3' for pGL2
272/+40 and pGL2 204/+40, respectively. The PCR fragments
were cloned into KpnI and BglII sites of the pGL3
basic luciferase vector.
Cell lines and culture systems. BJAB is an EBV-negative, human B-cell line obtained from Elliott Kieff (69). The B95-8 cells are singly infected with type I EBV (46, 47). The LCL-1 cell line was created in our lab by infection of primary human B lymphocytes with EBV (14). P3HR-1, an EBV-positive cell line from which portions of the EBNALP and EBNA2 open reading frames have been deleted, expresses low levels of LMP1 (59). BC-1 and BC-2, EBV- and KSHV-coinfected cell lines derived from patients with BCBLs, were purchased from the American Type Culture Collection (8, 24). The BJAB, P3HR-1, B95-8, and LCL-1 cell lines were grown in RPMI 1640 (Gibco-BRL Life Technologies Inc.) supplemented with 10% fetal bovine serum (Gemini Bioproducts Inc.), 2 mM L-glutamine, gentamicin (20 µg/ml), and penicillin-streptomycin (5 U/ml and 5 µg/ml, respectively). BC-1 and BC-2 cells were cultured as described above but in medium supplemented with 20% fetal bovine serum. Human embryonic kidney (HEK) 293 cells were grown in Dulbecco's modified Eagle medium (Gibco-BRL Life Technologies Inc.) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, gentamicin (20 µg/ml), and penicillin-streptomycin (5 U/ml and 5 µg/ml, respectively).
Transfections and luciferase assays. BJAB, P3HR-1, or 293 cells (107) were transfected with 5-µg quantities of the various promoter constructs or vector and increasing concentrations (5, 10, 15, or 20 µg) of the pCDNA3 LANA clones in 400 µl of RPMI 1640 (BJAB cells) or Dulbecco's modified Eagle medium (293 cells) containing 10% fetal bovine serum, using an electroporator at 210 V and 975 µF. Vector DNA was added to equalize the total amount of DNA used in all transfections, and its levels were normalized for transfection efficiency. After electroporation, cells were resuspended in 10 ml of complete medium, as described above, in 100-mm-diameter plates and incubated at 37°C for 20 h. Cells were harvested and then washed once with phosphate-buffered saline (PBS) and resuspended in the appropriate buffer. For luciferase assays, the cells were resuspended in 200 µl of 1× Reporter Lysis Buffer (Promega Inc.), placed in a dry ice-isopropanol bath for 5 min, thawed, vortexed for 15 s, and then centrifuged at 13,000 rpm for 20 s. Samples (45 µl each) were immediately combined with luciferase substrate in an Optocomp1 luminometer (MGM Instruments, Inc.) and counted for 10 s to determine the relative light units (RLU). The basic pGL vector alone was used in these assays to determine the ability of LANA to activate the vector alone. All assays were done in triplicate and repeated multiple times.
Western blot assays. Cells were collected by centrifugation and washed in 5 ml of PBS. The pellet was resuspended in an appropriate volume of sodium dodecyl sulfate (SDS) lysis buffer (57); lysates were analyzed by fractionation in SDS-6%, 8%, or 10% polyacrylamide gels for detection of LANA, EBNA2, and LMP1, respectively, and then transferred to 0.45-µm-pore-size nitrocellulose membranes. Membranes were blocked in 5% milk for 1 h at room temperature, rinsed three times with TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20), and then washed three times (5 min each) with TBST. The membranes were then incubated with the specific primary antibody overnight at 4°C before being put through the same washing regimen. The blots were then incubated with the appropriate secondary antibody for 1 h at room temperature and subsequently washed with TBST. Specific signals were detected by enhanced chemiluminescence as per the manufacturer's instructions (Amersham Pharmacia Biotech). LANA was detected with human polyclonal serum (diluted 1:50 in PBS supplemented with 1 mM sodium azide) followed by a protein A-horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech; 1:5,000 dilution). Hybridoma cell lines producing monoclonal antibodies PE2 and S12 (diluted 1:2 in hybridoma supernatant) were used to detect EBNA2 and LMP1, respectively, after which was added horseradish peroxidase-conjugated anti-mouse antibody (1:2,500 dilution). The proteins were then detected by enhanced chemiluminescence as suggested by the manufacturer (Amersham Pharmacia Biotech).
Immunofluorescence analysis.
Cells were washed twice in PBS
and resuspended at a density of 106 in 20 µl of
PBS. A 1-µl aliquot of the cells was placed on a slide and allowed to
air dry for 10 min. Slides were then fixed in precooled
acetone-methanol (1:1, vol/vol) at 20°C for 10 min and were allowed
to air dry. Cells were blocked with 20% goat serum for 30 min at room
temperature. To detect LANA, a polyclonal antibody was used at a 1:250
dilution in PBS for 1.5 h at room temperature. Four 5-min washes
with PBS were performed before addition of the secondary antibody, a
fluorescein isothiocyanate-conjugated goat anti-human antibody used at
a dilution of 1:1,000. Cells were incubated with the secondary antibody
for 1 h at room temperature. After being washed multiple times,
cells were mounted and visualized with an Olympus BX60 fluorescence
microscope and captured using the Espirit program, VI.2.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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LANA transactivates the EBV major LMP1-512/+72 and C latent promoters in the human BJAB cell line. In the coinfected BCBL cell lines, LANA is constitutively expressed. Therefore, we examined the effect of LANA on regulation of gene expression from the two major EBV promoters, Cp and LMP1p, in a B-cell background. This analysis in the BJAB cell line would more closely mimic the observed phenomenon in BCBL, which is derived from a B-cell line (4, 8). Cp is the major transcriptional promoter for the EBNA antigens, and LMP1, the viral oncoprotein critical for mediating EBV transformation of primary B lymphocytes, is transcribed from the LMP1 promoter (37, 56). Because LMP1 is expressed in the PEL cell lines, we wanted to compare the effects of LANA on the LMP1 promoter with those on the other major EBV latent promoter, Cp, which also known to be activated by EBNA2 (80).
To investigate LANA's ability to act as a transactivator of these promoters, the promoter-luciferase reporter constructs and the LANA expression constructs were transiently transfected, after which luciferase assays were performed. The results of these experiments demonstrate that LANA is a potent activator of the major C promoter, working in a dose-dependent manner (Fig. 2A). LANA upregulated the multimerized EBV Cp 15-fold compared with the promoter-only control. The effects were clearly dose responsive, with increasing amounts of LANA resulting in increased levels of transactivation. In this assay, the EBNA2 transactivator, which is known to activate Cp, activated at levels 10- to 11-fold above those for the promoter alone and therefore was used as a positive control (Fig. 2).
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LANA transactivates the EBV major LMP1-512/+72 and C latent
promoters in the HEK 293 fibroblast cell line.
To further
elaborate on the significance of the results obtained with BJAB cells,
we determined the effects of LANA in another human cell line, HEK 293. Similarly, we showed that LANA could upregulate the major EBV C
promoter in HEK 293 fibroblast cells. However, the effects were less
striking than those obtained for BJAB cells. LANA consistently
activated Cp three- to fourfold in HEK 293 cells compared with the
vector-only control (Fig. 3A). These
values were approximately threefold lower than those obtained with BJAB
cells. In HEK 293 cells, LANA activation of the 512/+72 LMP1 promoter
was greater than that with Cp (compare Fig. 3A and B), with the level
of activation being three- to sixfold higher than that of the
promoter-only control (Fig. 3B). Moreover, the results were
dramatically different from those for BJAB cells. A sevenfold reduction
in LANA-dependent activation was seen in HEK 293 compared with BJAB
cells, suggesting that LANA is a stronger activator of EBV promoters in
B cells than in 293 cells.
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LANA is expressed and localizes to the nucleus when transiently
transfected in the human cell lines BJAB and HEK 293.
To determine
if transiently transfected LANA was expressed and localized to the
nucleus in a fashion similar to that seen in KSHV-infected B cells, we
performed Western blot and immunofluorescence assays. Cells harvested
20 h after transient transfection and tested by Western blot and
immunofluorescence analyses indicated that LANA was expressed. In
addition, BJAB cells transfected with LANA had the characteristic
punctate pattern, similar to the staining pattern seen in the nucleus
of the KSHV-positive cell line BC-3 (Fig.
4). Similarly, in 293 cells, LANA
staining in the nucleus was punctate compared with that seen in BJAB
cells. The BC-3 KSHV-positive cell line was used as a control for LANA
signal in the nucleus (Fig. 4). The level of detection in the
transfected cells was similar to that seen in BC-3 (compare Fig. 4A, B,
and C). However, the LANA signal was slightly less punctate, although
the nuclear staining in specific regions was clearly seen. This may
have been an effect of overexpression and localization when a larger
amount of LANA was introduced transiently in cells compared with the amount used in the KSHV-infected cell line BC-3. No specific signals were seen for LANA in the cells transfected with vector alone (Fig. 4).
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LANA was obtained from polyclonal rabbit serum made against the
carboxy-terminal 300 amino acids of the LANA protein.
The truncated 204/+40 LMP1 promoter element is highly upregulated
by LANA in both BJAB and HEK 293 human cell lines.
To determine the
specific regions of LMP1p involved in LANA-mediated transactivation of
this promoter, two LMP1p reporter truncations were constructed; one had
a deletion of the AML1 binding site and the leftmost of the
RBP-J binding sites (272/+40 construct), and the second construct
had deletions of the AML1 binding site and both RBP-J sites (Fig.
5). The smaller construct, 204/+40, contains PU.1, Sp1, and ATF/CRE-responsive target sequences (30, 66). These sites were determined using a motif search program (TFSEARCH, version 1.3). These constructs were cotransfected in BJAB cells, and LANA and luciferase assays were performed.
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512/+40, 272/+40, and 204/+40 constructs) compared
with the promoter alone in 293 and BJAB cells (Fig.
6). Surprisingly, 272/+40, containing a
single RBP-J binding site, activated to lower levels than did
512/+40 and 204/+40 in BJAB cells. It is possible that the
repressive effects of RBP-J tempered the increased activation
mediated through the other downstream factors.
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272/+40 levels in BJAB cells
(Fig. 6). These results suggest that although LANA activates the
different truncated promoter elements to similar levels, the overall
transcriptional activity of each promoter element can differ in
specific cell lines.
The glutamic acid-rich central portion and carboxy terminus of LANA activate transcription of the LMP1 promoter. Based on the secondary structure of LANA, specific motifs suggest that the central glutamic acid-rich region, which is similar to that found in known transcription factors of the c-Fos/c-Jun family and other viral transcription factors, including EBNA3A and EBNA3C, can function as an activator of transcription (58, 77, 86). Additionally, the carboxy-terminal 400 amino acids of LANA contains a leucine zipper motif that is potentially involved in homo- or heterodimerization with other transcription factors (35, 52).
Specific domains of LANA (Fig. 1) containing the amino terminus (amino acids 1 to 435), the central glutamic acid-rich domain (amino acids 301 to 942), or the carboxy terminus (amino acids 762 to 1162) were cloned into expression vectors and tested in these transient reporter assays. As expected, LANA activated the512/+40 LMP1 promoter 10-fold over
the promoter alone control. The level of activation obtained with the
amino-terminal LANA truncation mutant (amino acids 1 to 435) was always
consistently about 50% (or less) of that seen with full-length LANA.
Surprisingly, the central LANA domain (amino acids 301 to 942) and the
carboxy-terminal LANA (amino acids 762 to 1162) constructs resulted in
activation levels 40-fold higher than that attained with the promoter
alone control and 4-fold higher than that observed for full-length LANA (Fig. 7A). The fact that the
central-domain and the carboxy-terminus constructs had similar levels
of activation suggests that these two regions may have a common motif,
since they overlap at amino acids 762 to 942 (Fig. 1). These results
suggested that a major activator of transcription might lie within
amino acids 762 to 942 of LANA and that the amino terminus, although
not completely devoid of activation functions, was clearly not the
major contributor to LANA's activity on the LMP1 promoter.
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The putative leucine zipper of LANA is not solely responsible for
mediating the transactivation functions of LANA.
The results of
the domain analysis indicated that the LANA region between amino acids
762 and 942 contains a leucine zipper motif which may be the
predominantly responsible for mediating activation of transcription
(35, 52). Therefore, we wanted to determine whether
constructs lacking the leucine zipper and the carboxy terminus and
constructs lacking but retaining the leucine zipper differed in their
ability to activate the LMP1 promoter. Both LANA truncation constructs
resulted in transcriptional activation of the 512/+40 LMP1 promoter.
Surprisingly, the LANA construct containing amino acids 1 to 756, which
lacks the putative leucine zipper, resulted in 20-fold activation, a
level similar to that seen with the LANA construct consisting of amino
acids 1 to 950, which contains the leucine zipper (Fig. 7B). These
results suggest that the glutamine-rich domain may contribute levels of activation similar to those of the leucine zipper domain and that the
domain downstream of the leucine zipper, amino acids 950 to 1162, may
also contribute to the overall activation activity encoded by LANA when
tested on the 512/+40 LMP1 reporter construct.
204/+40 construct had the highest activity, we wanted to
determine whether the effects on LANA domains would be more pronounced,
and therefore more clearly delineated, when tested on this smaller
construct. As described above, the LANA constructs consisting of amino
acids 1 to 435, 301 to 942, or 762 to 1162 were tested on the 204/+40
LMP1 reporter construct. In this assay, the construct which expresses
the glutamic acid-rich central domain and the leucine zipper had the
highest level of activity (Fig. 7C), about twofold greater than that
seen for the carboxy terminus lacking the glutamic acid-rich central
domain but containing the leucine zipper. When the constructs
consisting of amino acids 1 to 756 or 1 to 950 of LANA, containing the
glutamic acid-rich region without and with the leucine zipper,
respectively, were tested on the 204/+40 reporter construct, the
results showed that the portion of LANA that contains the glutamic
acid-rich region and the leucine zipper had an approximately twofold
higher level of activity than the construct with the glutamic acid-rich region when compared with the activity of the promoter alone (Fig. 7D).
These results suggest that although the glutamic acid-rich region makes
the greatest contribution to the activity of the LMP1 promoter, the
leucine zipper and the carboxy terminus of LANA are important and
contribute to the overall transcriptional activation of the LMP1
promoter seen with LANA. Immunofluorescence analysis indicated that
these LANA-truncated constructs all localized to the nucleus in BJAB
cells (Fig. 7E).
Western blot analysis shows that LMP1 is expressed in the
coinfected BC-1 and BC-2 cell lines expressing LANA.
As previously
shown, LANA is expressed in the coinfected BCBLs BC-1 and BC-2. In the
same cell lines, LMP1 is also expressed (5). We wanted to
show that the expression of LMP1 was maintained in these cell lines in
the absence of EBNA2 expression even after prolonged passage of these
cultures. BC-1 and BC-2 cell lines coinfected with KSHV and EBV were
passaged in culture over a 24-month period. From these
long-term-passage cultures, lysates were obtained and analyzed by
Western blotting for latent antigen expression. LANA was clearly
expressed in both cell lines (Fig. 8C);
however, EBNA2 was not detected (Fig. 8B). Again, LMP1 was expressed,
as evidenced in lanes 4 and 5 of Fig. 8A. LMP1 was also expressed in
the positive-control cell lines B95-8 and LCL-1. Interestingly, the
D1LMP1 signal seen in the B95-8 cell line was also seen in the
coinfected cell lines. These results corroborate the previous data
suggesting that LANA may contribute to activation of the EBV major
latent LMP1 promoter.
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Exogenously expressed LANA upregulates LMP1 in transiently
transfected P3HR-1 cells.
To determine if the levels of LMP1
change when LANA is expressed from a heterologous promoter, we
transfected an EBV-positive cell line exhibiting low levels of LMP1
expression to determine whether increased expression would occur when
the promoter was transactivated by LANA. P3HR-1 cells
(107) were transfected with increasing amounts of
LANA, and the transfected cells were harvested and analyzed for LMP1 by
Western blotting with a monoclonal antibody (S12) specific for LMP1.
The results demonstrated that LMP1 levels increased with increasing
amounts of LANA transfected (Fig. 9A).
Vector-alone controls with no LANA exhibited relatively low levels of
LMP1 in P3HR-1, as was expected (Fig. 9A, lane 4). It should be noted
that no change in levels of EBNA1 was seen by Western blot analysis.
Moreover, EBNA3C was not detected, suggesting that any effects on Cp in
the background of an endogenous genome were not detectable (data not
shown). This is consistent with the lower levels of Cp activation
observed in the reporter assays. The weak background bands evident in
the EBV-negative BJAB lane are nonspecific. Figure 9B shows a
cross-reactive band with equivalent intensities in all lanes,
indicating that similar levels of protein were loaded in all lanes.
Figure 9C is a Western blot showing that the levels of LANA increased
with increasing amounts of LANA transfected, as expected. The results from this assay suggest that LANA expressed from a heterologous promoter can upregulate LMP1 levels in the context of the EBV genomic,
native promoter elements.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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LANA is consistently detected in all KSHV-infected cells as well as in cells coinfected with both KSHV and EBV (7, 10). Since LMP1 is expressed in these cells in the absence of EBNA2, LANA may be capable of performing functions typically associated with the EBNA family of proteins encoded by EBV because of its localization in the nucleus, its restriction to the latent phase of infection of KSHV, and the apparent structural similarities among these proteins (5, 24, 35, 50, 71). LANA has already been shown to function, like EBNA1, by tethering the KSHV genome to chromosomal DNA, thereby facilitating viral episome persistence (2, 13). Also, LANA was shown to function as a transcriptional regulator in a number of studies, regulating KSHV and cellular promoters (38, 40, 51, 53, 63). Additionally, LANA has structural features typical of transcriptional activators, namely the central region containing repetitive blocks of acidic amino acids as well as a putative leucine zipper (35, 52). In fact, LANA has recently been shown to act as a transcriptional repressor and activator (38, 40, 63). These findings, as well as our inability to detect EBNA2 and other important EBV proteins in coinfected cells, led us to investigate LANA's potential role as a transcriptional activator of the EBV latent promoters in the case of KSHV-EBV coinfection.
By performing transient reporter assays, we have shown that LANA is an efficient transactivator of the two major latent promoters of EBV. Our experiments demonstrate that LANA can function similarly to EBNA2 in upregulating these promoters (24, 38). A recent study demonstrated that when LANA was cotransfected with EBNA2 and a Cp-chloramphenicol acetyltransferase reporter, LANA modulated the ability of EBNA2 to transactivate this promoter and LANA alone had no affect on Cp-chloramphenicol acetyltransferase activity (38). This is in contrast to our results, which demonstrate that LANA in fact can activate this promoter in two human cell lines. There are many experimental differences that could account for this discrepancy, including the cell lines utilized in the assay, the choice of reporters, and the amount of LANA expressed in these assays. Additionally, the effect on Cp by LANA in coinfected cells may be masked by expression of other viral or cellular proteins involved in viral gene regulation.
LANA's ability to transactivate the EBV LMP1 promoter triggers important questions about the cross talk between KSHV and EBV in the case of coinfected BCBL cells. Although EBNA2 has not been detected in any EBV- and KSHV-coinfected BCBL, LMP1 is consistently present in these cells, as documented in two separate reports (5, 24). Also, it should be noted that in another study LMP1 expression was not detected as is expected for a type I latency program (71). Since EBNA2 is the primary transactivator of the LMP1 promoter in EBV latency, it seems likely that another viral or cellular transactivator is involved in LMP1 upregulation. Therefore, like EBNA2, LANA can function as a transactivator of LMP1p in the case of KSHV-EBV coinfection, albeit through potentially separate mechanisms associated with only a portion of the overall role of LANA in these cell lines.
The fact that the basal activity of the truncated LMP1 promoters
increased with the deletion of upstream elements that include two
CBF1/RBP-J consensus sites corresponds with the fact that RBP-J
is a known repressor of transcription (21, 23, 25, 89).
EBNA2 interacts with RBP-J as well as other cellular proteins that
tether to the LMP1 promoter. The other significant factors involved in
this mechanism of activation are the PU.1 factor (a member of the Ets
family), an ATF/CRE, and the Sp1 transcription factor (31,
66).
Despite the ability of LANA to directly bind DNA to perform functions
associated with episomal maintenance (2, 13), it is
possible that LANA also utilizes its ability to associate with cellular
proteins to transactivate EBV LMP1p as well as other viral and cellular
promoters (38, 40). Whether LANA is directly binding to
the LMP1 promoter or interacting with cellular proteins, the region of
the promoter downstream of 204, shown to have enhanced activity, is
likely to contain sequences targeted by known potent activators of
transcription, such as ATF/CRE and Sp1 (31, 66, 87). Three
binding sites known to be important for EBNA2-mediated transactivation
of LMP1
namely, those for PU.1, ATF/CRE, and Sp1are all located
within this region (31, 66). Further truncations and
mutagenesis of LMP1p will be performed and similar experiments will be
done to determine the sequences required for LANA to efficiently transactivate the LMP1 promoter. Performing electrophoretic mobility shift assays with LANA and these cellular factors having target sequences within this region would provide clues to whether LANA can
directly complex with these factors bound to their cognate sequences.
Immunoprecipitation experiments will demonstrate their associations in
the KSHV- and EBV-coinfected cells.
To better characterize the region(s) of LANA responsible for mediating
transactivation of the EBV LMP1 promoter, we tested a number of LANA
constructs lacking specific domains of LANA in the transient reporter
assays. The amino terminus of LANA has minimal activity on EBV LMP1p,
while the constructs containing the central domain or the carboxy
terminus were potent activators of transcription. In comparing
constructs consisting of LANA amino acids 1 to 756 or 1 to 950, we
demonstrated that the putative leucine zipper domain contributes only
partially to the upregulation of the LMP1 promoter. There was little
difference between the LANA amino acid 301 to 942 and amino acid 762 to
1162 constructs, or between the constructs containing amino acids 1 to
756 or 1 to 950, with regard to 512/+40 promoter activation. The
constructs lacking the amino-terminal 301 amino acids consistently had
levels of activation higher than those lacking the region downstream of
the leucine zipper domain. This might imply that the amino terminus
contains repressive elements which may function in balancing the
regulatory activities of LANA in these coinfected cell lines. Different
LANA constructs will be made to further elucidate the specific regions
of the protein responsible for transactivation of the LMP1 promoter. It
should be noted that the central glutamic acid-rich domain had a
significantly higher level of activation when tested on the 204/+40
reporter construct. This suggests that the amino acid 301 to 942 domain
complexes with activators of transcription and is not regulated or
modulated by other cellular factors which associate with the domain
downstream of amino acid 950.
It is clear that LANA can upregulate LMP1p in the context of the virus. Our data demonstrate that LMP1 is consistently upregulated with increased levels of LANA exogenously expressed from a heterologous promoter. Therefore, LANA is capable of activating LMP1 at the level of its native promoter. This suggests a true transcription-regulatory function for LANA in affecting an EBV major essential latent promoter in coinfected PELs.
The accumulated data are beginning to suggest that proteins encoded by KSHV and EBV have the ability to regulate gene expression and have downstream oncogenic effects on each other in coinfected PEL cell lines. It appears that LANA is able to partially facilitate the role of EBNA2 as a transactivator of EBV latent promoters in EBV- and KSHV-coinfected lymphomas.
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ACKNOWLEDGMENTS |
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We thank Elliott Kieff for the BJAB cell line and Jon Aster and Jeffrey Sklar for the HEK 293 cell line. Joonho Choe kindly provided the amino acid 1 to 756 and amino acid 1 to 950 LANA constructs, and Elliott Kieff and Jeff Lin provided the pGL-LMP1-512/+40 promoter construct. We would thank Vojo Deretic and Jens Poschet for providing assistance and for use of the BX60 fluorescence microscope.
This work was supported by grants from the National Institutes of Health (NCI CA72150-01 to E.S.R.) and the Lymphoma and Leukemia Society of America. E.S.R. is a Scholar of the Lymphoma and Leukemia Society. M.A.C. is a fellow of the Lady Tata Memorial Trust and is supported by Medical Scientist Training Program grant T32 GM07863 to the University of Michigan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology and Comprehensive Cancer and Geriatrics Center, University of Michigan Medical Center, 3217 CCGC Building, Ann Arbor, MI 48109-0934. Phone: (734) 647-7296. Fax: (734) 764-3562. E-mail: esrobert{at}umich.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, October 2001, p. 9446-9457, Vol. 75, No. 19
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.19.9446-9457.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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