From the The Austin Research Institute, Austin
Hospital, Studley Road, Heidelberg 3084, Victoria, Australia and the
¶ Department of Microbiology and Immunology, Eastern Virginia
Medical School, Norfolk, Virginia 23501
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ABSTRACT |
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IFI 16 is a member of a family of interferon-inducible proteins, including the human MNDA (myeloid nuclear differentiation antigen), the recently identified AIM-2 (absent in melanoma), and the homologous murine molecules, p202, p204, and D3. IFI 16 contains a domain at the amino terminus capable of binding double-stranded DNA and a bipartite nuclear localization signal. No molecular or biological function has been assigned to any of the human family members, although a role in transcription regulation has been proposed. In the present study, we show IFI 16 fused to the GAL4 DNA binding domain can function as a transcriptional repressor. IFI 16-mediated repression is not dependent on the position or distance of IFI 16 binding, relative to the site of transcription initiation, and it can significantly repress when only one GAL4 DNA element is present in the promoter. We mapped the transcriptional repression domains to the 200 amino acid repeat regions common to all human and mouse family members. We also demonstrate that wild type IFI 16 can repress transcription of a reporter gene containing the minimal promoter region of the human cytomegalovirus UL54 gene. Thus, IFI 16 is a transcriptional repressor, with a modular structure typical of many known transcription regulators.
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INTRODUCTION |
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IFI 16 is a human protein with expression restricted to
fibroblasts, and specific hemopoietic cells such as lymphocytes and subsets of myeloid progenitor
cells.1 IFI 16 mRNA and
protein are absent from myeloid leukemia cells lines such as HL-60,
K562, and U937, but are strongly induced by
IFN2 and
, retinoic
acid, and Me2SO, agents that drive these cells to
differentiate along the myeloid lineage (2, 3). IFI 16 belongs to a
family of homologous IFN-inducible human and mouse proteins known as
the HIN-200 family (hemopoietic IFN-inducible Nuclear/200-amino acid repeat), each composed of at least
one common 200-amino acid motif of unknown function (4). The related members of this family identified thus far include human MNDA (myeloid
cell nuclear differentiation antigen) (5, 6), the recently identified
AIM 2 molecule (7), and the murine p202, p204, and D3 proteins (8).
Biochemical characterization of IFI 16 showed it to be localized to the
nucleus and indicated that the amino-terminal 159 amino acids were
necessary and sufficient to bind double-stranded DNA under
physiological conditions (3); however, the sequence specificity of this
interaction has not been determined to date. Similarly, the related
proteins, MNDA (5), p202 (9), and p204 (10) can also bind DNA and are
detected in the nucleus. The tightly regulated hemopoietic expression
of IFI 16, coupled with its biochemical features, led to the hypothesis
that IFI 16 may have a role in transcription regulation. This postulate was subsequently strengthened by the findings that p202 could interact
with the transcriptional regulatory proteins p53 (11), pRb (12), E2F
(13), c-Jun and c-Fos and NFB (14). Binding of p202 to p53, E2F, or
AP-1 family members impairs sequence-specific DNA binding by these
transcription factors, resulting in a loss of transcriptional activity
(11, 13, 14). However, no evidence exists for an "active" role in
transcription regulation by any members of the HIN-200 family.
The biochemical characteristics of IFI 16, its lineage-specific expression in hemopoietic cells, and induction in response to factors that drive cellular differentiation suggest a possible role in myelopoiesis. The expression and/or activation of certain transcription factors has been shown to induce the maturation of different hemopoietic lineages (15-17). For example, expression of the transcription factor GATA-1 is necessary for erythroid development (15), whereas PU.1 and Ikaros have been shown to be required for myelopoiesis (16) and T and B cell commitment (17), respectively. It is also clear, however, that lineage- and stage-specific transcription factors can function in combination to direct accurate hemopoietic development (18, 19). Whether IFI 16 is necessary and/or sufficient to regulate hemopoietic differentiation in a manner similar to factors such as GATA-1 or PU.1 remains unclear, but as a first step we set out to determine a possible role for IFI 16 in transcriptional regulation.
We chose to study the putative transcription modulating activity of IFI 16 in two systems. First, we expressed IFI 16 as a fusion protein with the yeast GAL4 DNA binding domain to direct IFI 16 to promoters containing consensus GAL4 DNA elements. We demonstrate that IFI 16 can function as a transcriptional repressor even when positioned approximately 1 kilobase pair downstream from the site of transcription initiation. The strength of repression by IFI 16 was illustrated in that binding to just one GAL4 site upstream of the promoter resulted in substantial activity. Significantly, we mapped the repression activity to either of the two 200-amino acid regions within IFI 16. These domains are common to all human and mouse family members, and thus it is possible that transcriptional repression may be a function of other related proteins.
Second, we examined whether IFI 16 plays a role in regulating expression of the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene. The minimal UL54 promoter contains a novel 8-bp inverted repeat element (IR1) that is capable of binding cellular proteins necessary for activation of UL54 by the viral immediate-early transcription factors IE72 and IE86 (20). Subsequent analysis showed that the transcription factor SP1, and IFI 16, can be copurified by DNA affinity chromatography using the region of the UL54 promoter containing the IR1 (21). We therefore tested for the effect of IFI 16 expression on a reporter gene containing the wild type UL54 minimal promoter. We showed that recruitment of wild type IFI 16 to the UL54 minimal promoter also results in transcriptional repression, indicating that the repression function of IFI 16 is not confined to the GAL4-IFI 16 fusion protein. This study is the first to assign a molecular function to any of the human members of this gene family and provides impetus to further examine the transcriptional regulatory function(s) and cellular differentiation properties of these molecules.
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MATERIALS AND METHODS |
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Plasmids--
pGAL4-tk-CAT and ptk-CAT were described previously
(22). pGAL4-IFI 16 was constructed by cloning full-length human IFI 16 cDNA in-frame into the pSG424 vector (23), which expresses the GAL4
(amino acids 1-147) DNA binding domain. pGAL4-IFI 16 1-125 was
produced by digesting IFI 16 cDNA with BamHI and
SacI and subcloning into the pSG424 vector digested with
BamHI and SacI. Plasmids pGAL4 IFI 16 1-201,
1-401, 1-518, 191-729, 191-400, and 509-729 were generated by
polymerase chain reaction using the IFI 16 forward primer and
appropriate reverse primers indicated below. Amplified DNA was digested
with BamHI and KpnI and subcloned into the
BamHI and KpnI sites of pSG424. The identities of
all constructs were confirmed by dideoxynucleotide sequence
analysis. The p5×GAL4UAS760,
p5×GAL4UAS+1000, 3×GAL4-tk-CAT, and 1×GAL4-tk-CAT
reporter constructs were described previously (24). The pCMV-IFI
16 expression plasmid was constructed by cloning full-length IFI 16 cDNA into the pRcCMV vector (Invitrogen), and the pPolCAT and
pIRMCAT reporter plasmids were described previously (20).
Oligonucleotides-- The sequences for oligonucleotides were as follows: IFI 16 forward primer, 5'-CCGGATCCTTATGTCTGTAAAGATG-3'; 201 reverse primer, 5'-GGGTACCTTCGAGAACATTTCTTCT-3'; 401 reverse primer, GGGTTACCGCTCTTGGGGTCATTGT-3'; 518 reverse primer, 5'-GGGTACCTGGGCACTGTCTTCTA-3'; 729 reverse primer, 5'- GGGTACCATCGTCAATGACATCCAG-3'; 191 forward primer, 5'-CCGGATCCAGGTAACTCCCAGAAGAA-3'; 509 forward primer, 5'-CCGGATCCCAAGACTGAAGACTGAA-3'.
Cells and Transfection-- HeLa or 293 cells were grown in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal calf serum. The cells were transfected by the calcium phosphate precipitation method (22). The total amount of DNA was adjusted with the plasmid pSP72 to be identical for each transfection. Cells were harvested 48 h after addition of the precipitate. All transfection assays were carried out with at least two independent DNA preparations and were repeated between three and five times.
CAT Assays-- Whole cell extracts were prepared from transfected cells. CAT activity was assayed as described (22) and quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Appropriate amounts of cell extracts were used to measure CAT activity to ensure that the assays were performed within linear range.
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting-- Nuclear lysates were prepared from cells transfected with various GAL4-IFI 16 effector plasmids by the Dignam method (25). Proteins were separated by electrophoresis through a 10% polyacrylamide gel, transferred onto Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA), and probed with monoclonal anti-GAL4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were incubated with a 1:10,000 dilution of horseradish peroxidase-coupled goat anti-mouse antibody (Dako, Carpinteria, CA), and immunoreactive proteins were visualized by ECL (Amersham Pharmacia Biotech, Buckinghamshire UK).
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RESULTS |
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IFI 16 Can Repress Transcription-- Earlier characterization of IFI 16 showed that this protein is specifically expressed in the nucleus and the amino-terminal 159 amino acids of IFI 16 could bind double-stranded DNA. These properties are characteristics of transcription factors, and thus we sought to determine a possible transcriptional regulatory role for IFI 16. Full-length IFI 16 was fused to the yeast GAL4 DNA binding domain (amino acids 1-147). This truncated form of GAL4 can bind with high affinity to the GAL4 consensus DNA sequence but has little or no effect on transcription (23). GAL4-IFI 16 and the GAL4-tk-CAT reporter plasmid, containing five copies of the GAL4 binding site arranged in tandem approximately 150 bp upstream of the CAT reporter gene, were cotransfected into HeLa cells, and the effect on CAT expression was determined. Expression of GAL4-IFI 16 resulted in a dose-dependent decrease in CAT expression with a maximal 5-fold decrease in CAT activity observed after transfection with 3 µg of GAL4-IFI 16 (Fig. 1, lanes 1-3). As a specificity control, GAL4 alone was cotransfected with GAL4-tk-CAT, and this resulted in a slight increase in CAT activity (Fig. 1, lanes 4 and 5). Thus, IFI 16 could efficiently repress CAT activity when bound upstream of the reporter gene. Cotransfection of GAL4-IFI 16 with the reporter plasmid tk-CAT, which is devoid of GAL4 binding elements, did not result in any change in CAT activity (Fig. 1, lanes 6 and 7). Similarly, expression of GAL4 did not effect CAT expression from the tk-CAT reporter plasmid (Fig. 1, lane 8). Similar results were also observed in human 293 cells and murine NIH 3T3 cells (data not shown), indicating that repression by IFI 16 was not cell type- or species-dependent.
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Repression by GAL4-IFI 16 Is Not Dependent on the Position of the
Binding Sites Relative to the Transcription Start Site--
We have
shown that GAL4-IFI 16 can repress transcription of a reporter gene
when the GAL4 binding sites are positioned approximately 150 bp
upstream of the cap site (Fig. 1). To determine whether GAL4-IFI 16 could function as a repressor when bound at a distance from the
thymidine kinase promoter, reporter constructs containing GAL4 sites
760 bp upstream of the cap site (5×GAL4UAS760) and 1000 bp downstream of the cap site (5×GAL4UAS+1000) were used.
GAL4-IFI 16 could repress transcription of a reporter gene when the
GAL4 binding sites were positioned 760 bp upstream of the transcription
initiation site (Fig. 2). This repression
was dose-dependent and reached a maximum of approximately
5-fold repression upon cotransfection of 3 µg of GAL4-IFI 16 plasmid
with the 5×GAL4UAS
760 reporter plasmid (Fig. 2,
lanes 1-3). No effect on this reporter plasmid was seen
upon expression of GAL4 DNA binding domain alone (Fig. 2, lanes
4 and 5). Thus, IFI 16 can function as a
transcriptional repressor when positioned at least 760 bp upstream of
the transcription initiation site. Similarly, GAL4-IFI 16 also
repressed CAT expression when the GAL4 binding sites were positioned
1000 bp downstream of the transcription start site (Fig. 2, lanes
6-8). Cotransfection of 1-3 µg of GAL4-IFI 16 with the
5×GAL4UAS+1000 reporter plasmid caused a similar decrease
in CAT activity as was seen with the GAL4-tk-CAT and
5×GAL4UAS
760 reporter plasmids. Repression by GAL4-IFI
16 was dependent on the GAL4-IFI 16 fusion protein being positioned at
the thymidine kinase promoter, because an effect on a reporter
construct without GAL4 binding sites was not observed (data not
shown).
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Effect of Multiple GAL4 DNA Binding Sites on Repression by GAL4-IFI 16-- Many transcriptional activators function synergistically when bound to multiple sites within a given promoter (26). To analyze possible synergistic repression by GAL4-IFI 16, the CAT reporter plasmids 5×GAL4-tk-CAT, 3×GAL4-tk-CAT, and 1×GAL4-tk-CAT containing 5, 3, and 1 GAL4 binding elements, respectively, approximately 150 bp upstream of the CAT gene were used. As shown in Fig. 1, GAL4-IFI 16 could significantly decrease CAT activity from a reporter plasmid containing five GAL4 sites (Fig. 3, lanes 1-3). GAL4-IFI 16 similarly repressed CAT expression from the 3×GAL4-tk-CAT reporter in a dose-dependent manner (Fig. 3, lanes 5-7). Significant repression by GAL4-IFI 16 on a plasmid containing only one GAL4 binding element was still observed, although 2-fold less than when five or three GAL4 binding sites were present in the reporter plasmid (Fig. 3, lanes 9-11). Thus GAL4-IFI 16 can still significantly repress transcription when only one GAL4 binding site is present in the promoter region upstream of CAT and does not appear to function in a synergistic manner.
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IFI 16 Contains Separate DNA Binding and Repression Domains-- To determine whether a specific region of IFI 16 was responsible for the observed repression function of GAL4-IFI 16, we produced COOH- and NH2-terminal deletion mutants of IFI 16 fused to the GAL4 DNA binding domain and assayed for repression activity using the GAL4-tk-CAT and tk-CAT reporter plasmids (Fig. 4A). As shown previously, full-length IFI 16 fused to GAL4 caused a dose-dependent decrease in CAT activity with a maximal 5-fold decrease observed upon transfection of 3 µg of GAL4-IFI 16 (Fig. 4B, lanes 2 and 3). To determine the contribution of the carboxyl terminus of IFI 16 to its repression function, deletion mutants were constructed by polymerase chain reaction. Cotransfection of GAL4-IFI 16(1-125) and GAL4-IFI 16(1-201) containing the IFI 16 amino-terminal 125 and 201 amino acids, respectively, did not significantly affect CAT expression (Fig. 4B, lanes 4-7). However GAL4-IFI 16 fusion proteins containing the 200-amino acid A repeat (GAL4-IFI 16(1-401)) or the 200-amino acid A repeat plus the hinge region (GAL4-IFI 16(1-518)) could clearly function as transcriptional repressors (Fig. 4B, lanes 8-11). Both of these fusion proteins mediated similar repression at the highest concentration of transfected effector plasmid to that seen with full-length IFI 16.
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Wild Type IFI 16 Can Repress Transcription-- To determine whether wild type IFI 16 can also repress transcription, we used the pPolCAT reporter plasmid containing the minimal promoter region of the HCMV DNA polymerase (UL54) gene (20) found to bind IFI 16 (21). The HCMV DNA polymerase promoter contains a novel 8-bp inverted repeat 1 element (IR1), and mutation of the wild type IR1 element abrogates UL54 promoter activity and abolishes binding of cellular protein(s) to the IR1 (20, 21). Cotransfection of the IFI 16 expression plasmid with pPOLCAT in 293 cells resulted in a dose-dependent decrease in CAT activity (Fig. 5A, lanes 2 and 3), whereas cotransfection with the empty CMV expression vector had no effect (Fig. 5A, lanes 4 and 5). We also tested for the effect of IFI 16 on the pIRMCAT reporter plasmid, which contains a 4-bp functional mutation in the IR1 (20). Cotransfection of CMV-IFI 16 or CMV alone with pIRMCAT resulted in little or no change CAT activity (Fig. 5B), indicating that a functional IR1 element is necessary for transcriptional repression by IFI 16.
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DISCUSSION |
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IFI 16 is a well characterized member of the HIN-200 family of
proteins, but up until now the molecular function(s) of this molecule
were unknown. Some of the physical characteristics of IFI 16, such as
its induction by and
IFN, its strong nuclear localization, and
its ability to bind to DNA through its amino terminus hinted at a
possible role in transcription regulation. In light of the
circumstantial evidence indicating a possible role of IFI 16 in
transcription, and because it had been shown that the related mouse
protein, p202, could modulate the activity of known transcription
factors (11-14), we set out to analyze the gene regulation effect of
IFI 16. We initially used the GAL4 model system, utilizing the
heterologous GAL4 DNA binding domain fused to IFI 16 as an effector
protein and GAL4-tk-CAT or tk-CAT reporter plasmids. This region of
GAL4 is transcriptionally inert, being neither capable of activating
nor repressing transcription. GAL4-IFI 16 could strongly repress
transcription of a reporter gene in a dose-dependent
manner, and this repression effect was dependent on IFI 16 being
tethered to DNA within the promoter region. The strength of IFI
16-mediated transcriptional repression was demonstrated in two separate
experiments. First, IFI 16 could specifically repress transcription
when bound approximately 760 bp upstream or 1000 bp downstream from the
transcription start site. The extent of repression was similar to that
seen when IFI 16 was bound only 150 bp upstream of the transcription
start site. This is in contrast to the transcriptional repressor WT1,
which in the same system shows greatly decreased repression activity
when positioned further upstream or downstream of the CAT reporter gene
(24). Second, GAL4-IFI 16 could significantly repress transcription
when only a single GAL4 binding element was present in the promoter
region. As GAL4 binds DNA as a dimer, this indicates that as few as two IFI 16 molecules positioned at a given promoter are sufficient to
negatively regulate gene transcription.
As with many transcriptional activators, proteins capable of repressing transcription are often modular proteins with distinct regions capable of binding DNA, interacting with other cellular and viral proteins, and actively repressing transcription (27). Proteins that actively repress are, by definition, thought not to act by competing with transcriptional activators for access to a particular DNA element, but function by direct interaction with components of the basal transcription machinery or transcriptional activators (27). As such, these proteins should be able to function as transcriptional repressors when fused to a heterologous DNA binding domain and brought to a promoter that contains the appropriate consensus DNA site.
IFI 16 joins a growing list of proteins capable of active repression. Some of these proteins such as YY1 (22) and WT1 (28) are capable of binding DNA in a sequence-specific manner, whereas others such as mSin3A and mSin3B (29, 30) and the yeast Tup 1/SSn6 (31) proteins interact with DNA-binding proteins and are thereby brought to a particular promoter. IFI 16 can bind to double-stranded DNA and could be eluted by similar salt elution protocols as those used for known sequence-specific transcription factors (3); however, specific DNA elements that can bind IFI 16 have yet to be identified and are currently under investigation. Recently it was shown that SP1 and IFI 16 can be copurified by DNA affinity chromatography using a region of the HCMV DNA polymerase (UL54) promoter necessary for gene activation by the HCMV immediate-early proteins IE72 and IE86 (21). Cotransfection of full-length IFI 16 and a CAT reporter gene containing the minimal wild type UL54 promoter results in repression by IFI 16, indicating that the repression results seen with GAL4-IFI 16 may also hold true when wild type IFI 16 is bound to DNA. It is presently unknown whether IFI 16 and SP1 physically interact. However, it has been shown that a wild type IR1 element within the minimal UL54 promoter is necessary for both SP1 binding (21) and IFI 16-mediated transcriptional repression as shown herein. In addition, activation of the UL54 promoter by HCMV immediate-early proteins requires binding of cellular proteins to the IR1 (20, 21), but IE-86 does not interact with DNA-bound SP1 (21). It is possible, therefore, that IE86 and/or IE72 can interact with IFI 16 or a complex consisting of IFI 16 and SP1 bound to the IR1 to activate the UL54 promoter. Studies to address this hypothesis are currently under way.
As with p202, IFI 16 can also bind to the transcriptional regulatory proteins p53 and pRb, both in vitro and in vivo,3 and might therefore also modulate the transcriptional activities of these and other gene regulatory proteins. The effect of IFI 16 binding to p53 and pRb is currently under investigation; however, both p53 and Rb are themselves capable of active repression (1, 32, 33), giving rise to the possibility that the mechanism of repression by IFI 16 may involve corepressors such as p53 and/or Rb.
To determine the region(s) of IFI 16 capable of actively repressing transcription, we performed deletion mutagenesis and fused the truncated portions of IFI 16 to GAL4. Consistent with IFI 16 being an active repressor, the amino-terminal, DNA binding region was incapable of affecting transcription when fused to GAL4. However a larger protein that also contained the first of the 200-amino acid repeat regions common to this family of proteins was capable of repressing transcription to a similar degree as seen with full-length IFI 16. This indicates that a repression domain of IFI 16 could reside within this 200-amino acid repeat. Expression of the A or B 200-amino acid repeat of IFI 16 fused to the GAL4 DNA binding domain was sufficient to induce transcriptional repression. However, expression of the carboxyl-terminal 539 amino acids (containing the A repeat, the B repeat, and the hinge region) fused to the GAL4 DNA binding domain did not result in transcriptional repression. This unexpected result could not be explained by poor expression or nuclear localization as GAL4-IFI 16(191-729) was expressed to a similar degree as the other fusion proteins used in this study. It is possible that removal of the amino-terminal 190 amino acids results in a conformational change deleterious for IFI 16's transcriptional repression function. Thus, repression by IFI 16 may be dependent on the conformation of the protein. This may be important given our recent findings that at least three different IFI 16 protein isoforms exist because of alternative RNA splicing.4 The splice variants encode proteins with a longer or shorter hinge region, thereby changing the distance between the A and B 200-amino acid repeats and possibly changing the conformation of the molecule.
The biological significance of the transcriptional repression function of IFI 16 is still unknown. However its proposed role in hemopoiesis and our recent finding that IFI 16 can suppress cell growth4 may indicate that IFI 16 could act in a similar manner to other transcription factors such as GATA-1 and PU.1, which control cell growth and differentiation by regulating key cellular genes (15, 16). In addition, the exciting finding that IFI 16 may regulate HCMV DNA polymerase expression leads to the hypothesis that IFI 16 may play a key role in HCMV infection. Clearly, the cellular genes whose expression may be regulated by IFI 16 need to be identified.
This study is the first to show a molecular function for a human member
of the HIN-200 family of proteins. Studies on the related murine
protein p202 have shown it capable of modulating the transcriptional
activities of well defined transcription factors such as E2F, p53,
c-Jun, and c-Fos by inhibiting binding to their consensus DNA elements
(11, 13, 19). However, p202 can also inhibit transcriptional activation
by NFB without any effect of the ability of NF
B to bind DNA (14).
Thus, ranscriptional repression may be a property of other HIN-200
family members, and studies to address this are currently being
undertaken.
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ACKNOWLEDGEMENTS |
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We thank Dr. Y. Shi and Dr. F Rauscher III for useful reagents and Drs. M. J. Smyth and S. M. Russell for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ CJ Martin Fellow of the National Health and Medical Research Council of Australia. To whom correspondence should be addressed. Tel.: 61-3-9287-0655; Fax: 61-3-9287-0600; E-mail: r.johnstone{at}ari.unimelb.edu.au.
Senior Research Fellow of the National Health and Medical
Research Council.
1 M. J. Dawson, N. J. Elwood, R. W. Johnstone, and J. A. Trapani, submitted for publication.
2 The abbreviations used are: IFN, interferon; pRb, retinoblastoma protein; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; HCMV, human cytomegalovirus; IR1, inverted repeat 1; bp, base pair(s).
3 R. W. Johnstone and J. A. Trapani, manuscript in preparation.
4 R. W. Johnstone and J. A. Trapani, unpublished observations.
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REFERENCES |
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