Institute of Biotechnology1 and Department of Biology2, National Cheng Kung University, Tainan 701, Taiwan
Author for correspondence: Hong-Hwa Chen. Fax +886 6 276 6505. e-mail hhchen{at}mail.ncku.edu.tw
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Abstract |
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Introduction |
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Hz-1 virus was the first invertebrate virus to be studied for temporal gene expression during productive and persistent virus infections (Chao et al., 1992 ). It replicates in the nuclei of infected cells and produces numerous virions during productive infection in most cells. Persistent infection is established in only a very small proportion (0·010·05%) of infected cells (Granados & Williams, 1986
; Lin et al., 1999
). During a productive infection, Hz-1 virus produces more than 100 different transcripts, but only one transcript is predominantly detected in a persistent infection, the persistence-associated transcript 1 (PAT1) (Chao et al., 1992
). PAT1 is also expressed as early as 2 h post-infection (p.i.) in productive infection, and its expression level remains constant up to 12 h p.i. (Chao et al., 1992
). It has been suggested that PAT1 is involved in the establishment of Hz-1 virus persistence (Chao et al., 1998
).
Previous studies showed that PAT1 is a transcript with several unique features (Chao et al., 1998 ). Sequence analysis of PAT1 reveals abundant direct and inverted repeats. No significant open reading frames (ORFs) can be detected in any reading frames. The lack of ORFs in the PAT1 sequence was confirmed by the fact that it is not associated with polysomes (Chao et al., 1998
). Furthermore, PAT1 is a nuclear RNA, as was concluded from subcellular fractionation and in situ hybridization (Chao et al., 1998
). The fact that the promoter sequence of PAT1 is adjacent to its transcription start site argues that PAT1 is not a spliced intron of a large transcript. In addition, the CAGT motif, an initiator element for early transcription of the baculovirus ie-1 gene (Pullen & Friesen, 1995
), is located at nucleotide (nt) +3 to +6 of the PAT1 transcript. All the abovementioned characteristics suggest that PAT1 does not encode a protein.
The minimal requirement of the PAT1 promoter has been determined by progressive deletions from the upstream region (Chao et al., 1998 ). Deletions up to nt -212 resulted in a threefold increase in promoter activity as compared to deletions up to nt -315. Deletions up to nt -158 showed similar levels of promoter activity to the deletion up to nt -212. A further deletion to nt -90 caused a sevenfold increase in promoter activity as compared to the promoter with a deletion to nt -315 (Chao et al., 1998
). These results suggested that both -315/-212 and -158/-90 regions exert negative regulatory effects.
In this report, we confirm that the -312/-90 region of the PAT1 promoter exerts an inhibitory effect on expression from the heterologous IE0 promoter of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The AcMNPV IE0 promoter was used as it has been well studied (Kovacs et al., 1991 ). Besides, both Hz-1 virus and baculovirus replicate in insect cells, and this lends strength to the fact that this study will be useful in understanding transcription in insect virusinsect cell systems. Our results show that the inhibitory effect is independent of its orientation towards the promoters. Computer-assisted analysis shows that two GATA elements are present in the -312/-90 region of the PAT1 promoter. GATA family transcription factors share one or two copies of a highly conserved zinc finger domain that binds with the core sequence WGATAR (W, A/T; R, A/G). Substitutions of the four core nucleotides in both GATA elements of the -312/-90 region eliminated its negative regulatory activity, suggesting that both GATA elements are involved in the inhibitory regulation.
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Methods |
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PCR and construction of plasmids.
Primers with XhoI or HindIII restriction enzyme sites at their 5' ends were used to amplify various PAT1 promoter regions: -727/-505, -312/-90, mut-312/-90, -312/mut-90 and -158/-90 (Table 1) from pHzEM, which contains the EcoRI M fragment of the Hz-1 genome harbouring the PAT1 coding region. The amplification reactions were performed with a thermocycler (Biometra) with 30 cycles of 94 °C for 40 s, 55 °C for 1 min, 72 °C for 2 min. The PCR-amplified fragments were purified after fractionation in low melting temperature agarose (Sea Plaque GTG, FMC Bio-Products), digested with XhoI or HindIII (NEB) and then ligated into the XhoI- or HindIII-linearized plasmid pTSV/IE0 containing a lacZ reporter gene under the control of a 589 bp fragment containing the IE0 promoter of AcMNPV (Lee et al., 1995
). The resulting plasmids, pTSV/IE0/-312/-90, pTSV/IE0/-312/-90R, pTSV/IE0/-727/-505, pTSV/IE0/-312/mut-90, pTSV/IE0/mut-312/-90 and pTSV/IE0/mut-312/mut-90 were sequenced to determine the direction of inserts. pTSV/IE0/-312/-90R is the construct containing the -312/-90 fragment in reverse orientation.
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-Galactosidase activity assay and CAT assay.
Cell lysates were prepared by three freezethaw cycles. For the determination of -galactosidase (
-gal) activity, cell lysates were incubated with a reaction mixture containing 25 mM Tris, pH 7·5, 125 mM NaCl, 2 mM MgCl2, 12 mM
-mercaptoethanol and 0·3 mM 4-methylumbelliferyl-
-D-galactoside at 37 °C for 30 min. To stop the reaction, TCA was added to the mixture and immediately chilled on ice. Subsequently, glycine carbonate reagent was added. The emitted fluorescence was detected by a minifluorometer (Hoefer, TKO 100). Statistical analysis was carried out using the MannWhitney Rank Test in the SigmaStat statistics software package (Jandel Scientific Corp.). CAT assays were carried out according to Nissen & Friesen (1989)
.
Preparation of nuclear extracts.
SF9 cells (2x107) were resuspended in ice-cold buffer A (15 mM KCl, 10 mM HEPES, pH 7·6, 2 mM MgCl2, 0·1 mM EDTA, 1 mM DTT, 0·1% NP40 and 0·5 mM PMSF) and the mixture was incubated on ice for 10 min. The nuclei were collected by centrifugation at 500 g for 10 min and resuspended in buffer B (1 M KCl, 25 mM HEPES, pH 7·6, 0·1 mM EDTA, 1 mM DTT and 0·5 mM PMSF). The suspension was incubated at 4 °C for 15 min and vortexed every 3 min. The lysate was centrifuged at 20800 g for 20 min (Eppendorf 5417R). The supernatant was transferred to a new tube and diluted with buffer C (20% glycerol, 25 mM HEPES, pH 7·6, 0·1 mM EDTA, 1 mM DTT and 0·5 mM PMSF) at a ratio of 1:3·75 (Inoue et al., 1994 ). The protein concentration in the nuclear extracts was determined using a Protein Assay kit (Bio-Rad).
Gel mobility shift assay and competition experiments.
These were performed according to Inoue et al. (1994) with some modifications. Both positive (+) and negative (-) strands of 20-mer oligonucleotides containing the GATA element (Table 1
) were synthesized, annealed and end-labelled with [
-32P]ATP by T4 polynucleotide kinase (NEB). Labelled probe (1 ng) was incubated with 5 µg nuclear extract in binding buffer (12 mM HEPESNaOH, pH 7·6, 4 mM TrisHCl, pH 7·6, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 12% glycerol and 2·5 mM PMSF) in the presence of 1 µg of poly dI-dC (Gibco BRL) for 30 min at room temperature. DNAprotein complexes were analysed on a 6% native polyacrylamide gel using 1x TBE electrophoresis buffer at 100 V for 6 h. Gels were then dried and autoradiographed. Competition experiments were performed as described by Inoue et al. (1994)
, except that labelled probe was incubated with competitor oligonucleotides prior to the addition of nuclear extract. The percentage of competition was determined by measuring band intensities using an image analyser (FLA-1000, Fujifilm).
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Results |
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To identify the protein-binding motifs within the -158/-90 and the -312/-212 regions, the sequences of these fragments were compared with the database of the Human Genome Program of Japan. Sequence analysis revealed that each fragment had one GATA element and binding motifs for transcription factors such as heat shock factor (HSF) (Sistonen et al., 1994 ; Morano & Thiele, 1999
), c-Myc and Cdx A (Margalit et al., 1993
; Frumkin et al., 1993
).
Analysis of the GATA elements in the -312/-90 region
Among the proteins that have binding motifs in the -312/-90 region, both HSF and c-Myc are positive regulators, and only the GATA-binding protein has been shown to have both positive and negative regulatory effects on gene expression (Orkin, 1992 ; Yang & Evans, 1995
). Thus, the GATA element was most likely to exert the negative effect on PAT1 promoter activity. Therefore, we investigated whether the GATA element was responsible for the observed gel-retarded complexes in EMSAs with the -158/-90 region. A 20-mer oligonucleotide duplex containing the GATA element based on the -158/-90 region (Table 1
) was used as a probe for EMSAs. A DNAprotein complex was detected with the GATA element probe (Fig. 3a
, lane 2). This complex was specific, since the binding decreased upon addition of 10- to 100-fold molar excess of the unlabelled GATA oligonucleotide (Fig. 3a
, lanes 46). However, the complex formation could not be outcompeted for by a 100-fold molar excess of non-specific competitor 7-4, a 20 bp fragment derived from the hhi1 gene of Hz-1 virus (Fig. 3a
, lane 7). These results suggest that a specific protein or proteins recognize the GATA element located within the -158/-90 region.
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To confirm that the GATA element was responsible for the observed DNAprotein interaction within the -158/-90 region, competition experiments were performed with fragment -158/-90 as probe and the 20-mer GATA element oligonucleotide as competitor. As indicated in Fig. 4, the amount of complex a formed with the -158/-90 fragment was reduced by 40% as measured by an image analyser using a 100-fold molar excess of GATA oligonucleotide as a specific competitor. A 200-fold molar excess of GATA oligonucleotide can compete for the complexes a, c, d and e, while complexes b and f were unaffected (Fig. 4
). Two extra complexes are visible, one minor and one major. The minor one is located between complex c and d, and the major one is located between complex f and the probe. These two complexes were not detected in previous results using the -158/-90 region as probe (Fig. 2a
). Occasionally, this occurred with different nuclear extract preparations. Besides, these two extra bands were not competed for even at a 200-fold molar excess of GATA oligonucleotide, suggesting that they are nonspecifically binding complexes.
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Discussion |
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Both positive and negative regulatory effects on the different genes have been identified for GATA-1 transcription factor (Orkin, 1992 ; Yang & Evans, 1995
). In mouse erythroleukaemia cells, GATA-1 can activate
-globin gene expression (Amrolia et al., 1995
), while it has a negative regulatory effect on the
-globin gene (Raich et al., 1995
). In human K562 cells, GATA-1 has a positive effect on the human ferrochelatase gene (Turgores et al., 1994
). Drosophila pannier, a GATA-1-like protein, plays a negative regulatory role on the achaete and scute genes involved in bristle formation (Ramain et al., 1993
).
Our report here demonstrates that two GATA elements were identified in nt -312/-90 of the PAT1 upstream promoter region of Hz-1 virus. This fragment exerted a negative regulatory effect on transcription from both the PAT1 promoter and the heterologous AcMNPV IE0 promoter. Either GATA element in the -312/-90 region was adequate for the inhibition of promoter activity. Both -315/-212 and -158/-90 regions contribute to the negative regulatory effects equally. Interestingly, we found that the negative regulatory effect of the -312/-90 fragment was independent of its orientation to the promoter, which resembles the situation found with the silencer in yeast and other organisms (Ogbourne & Antalis, 1999 ).
There are six complexes (complex a to f) formed with the -158/-90 region (Fig. 2a). Among them, complex a is most likely related to the GATA oligonucleotide, since it was reduced by 40% using a 100-fold molar excess of GATA oligonucleotide, and complexes c, d and e were competed for with 200-fold molar excess of GATA oligo, while complexes b and f were unaffected. It is possible that complexes c, d and e might originate from proteinprotein interactions between cellular proteins that recognize sequences within the -158/-90 region and the GATAprotein complex, or from the cellular proteins binding to the GATAprotein complex.
The GATA motif has been shown to be present in several baculovirus genes for binding of the insect GATA-like proteins. The promoter of PE38, a very early gene of AcMNPV encoding transcription factors containing two DNA-binding motifs, a zinc finger and a leucine zipper, contains a GATA motif recognized by Spodoptera frugiperda nuclear factor 1. Besides, computer analysis also showed that the A/TGATAT/C sequences are present in promoters of the immediate early genes of baculovirus, including 35K, ME53 and IEN of AcMNPV and IE1 of Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) (Krappa et al., 1992 ). It has been suggested that the insect GATA-binding proteins can recognize the GATA motif of 35K and ME53 promoters. However, point mutations in the GATA-1-binding site of PE38 did not affect its promoter activity, suggesting that GATA-1 is not essential for the expression of the PE38 gene (Krappa et al., 1992
). In contrast, the removal of GATA abolishes the binding of host transcription factor to the gp64 gene of OpMNPV and reduces transcription activation, suggesting that the GATA-1-binding sequence has a regulatory role on gp64 (Kogan & Blissard, 1994
).
In conclusion, two GATA elements on the -312/-90 region of the PAT1 promoter are related to the negative regulation effect of this fragment. The identification of these elements facilitates the understanding of the regulation of PAT1 transcription.
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Acknowledgments |
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References |
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Chao, Y.-C., Hamblin, M. & Wood, H. A.(1990). Physical map of Hz-1 baculovirus genome from standard and defective interfering particles. Journal of General Virology 71, 1265-1270.
Chao, Y. C., Wood, H. A., Chang, C. Y., Lee, H. J., Shen, W. C. & Lee, H. T.(1992). Differential expression of Hz-1 baculovirus genes during productive and persistent viral infections. Journal of Virology 66, 1442-1448.[Abstract]
Chao, Y.-C., Lee, S. T., Chang, M. C., Chen, H. H., Chen, S. S., Wu, T. Y., Liu, F. H., Hsu, E. L. & Hou, R. F.(1998). A 2·9 kb non-coding nuclear RNA functions in the establishment of persistent Hz-1 viral infection. Journal of Virology 72, 2233-2245.
Drevet, J. R., Skeiky, Y. A. W. & Iatrous, K.(1994). GATA type zinc finger motif-containing sequences and chorion gene transcription factors of the silkworm Bombyx mori. Journal of Biological Chemistry 269, 10660-10667.
Drevet, J. R., Swevers, L. & Iatrous, K.(1995). Developmental regulation of a silkworm gene encoding multiple GATA-type transcription factors by alternative splicing. Journal of Molecular Biology 246, 43-53.[Medline]
Frumkin, A., Haffiner, R., Shapira, E., Tarcic, N., Gruenbaum, Y. & Fainsod, A.(1993). The chicken CdxA homeobox gene and axial positioning during gastrulation. Development 118, 553-562.
Granados, R. R. & Williams, K. A.(1986). In vivo infection and replication of baculoviruses. In The Biology of Baculoviruses , pp. 159-175. Edited by R. R. Granados & B. A. Federici. Boca Raton:CRC Press.
Granados, R. R., Nguyen, T. & Cato, B.(1978). An insect cell line persistently infected with a baculovirus-like particle. Intervirology 10, 309-317.[Medline]
Huang, Y. S., Hedberg, M. & Kawanish, C. Y.(1982). Characterization of the DNA of a nonoccluded baculovirus, Hz-1 V. Journal of Virology 43, 174-181.
Inoue, N., Dambaugh, T. R., Rapp, J. C. & Pellett, P. E.(1994). Alphaherpesvirus origin-binding protein homolog encoded by human herpesvirus 6B, a betaherpesvirus, binds to nucleotide sequences that are similar to ori regions of alphaherpesvirus. Journal of Virology 68, 4126-4136.[Abstract]
Kogan, P. H. & Blissard, G. W.(1994). A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. Journal of Virology 68, 813-822.[Abstract]
Kovacs, G. R., Guarino, L. A. & Summers, M. D.(1991). Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus. Journal of Virology 65, 5281-5288.[Medline]
Krappa, R., Behn-Krappa, A., Jahnel, F., Doerfler, W. & Knebel-Morsdorf, D.(1992). Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein. Journal of Virology 66, 3494-3503.[Abstract]
Lee, S. T., Yu, S. M., Hsu, E. L. & Chao, Y. C.(1995). Identification of a very early promoter of insect Hz-1 virus using a novel dual-expression shuttle vector. Nucleic Acids Research 23, 4683-4689.[Abstract]
Lin, W. H., Huang, L. H., Yeh, J. Y., Lehrach, H., Sun, Y. H. & Tsai, S. F.(1995). Expression of a Drosophila GATA transcription factor in multiple tissues in the developing embryos. Journal of Biological Chemistry 270, 25150-25158.
Lin, C. L., Lee, J. C., Chen, S. S., Wood, H. A., Li, M. L., Li, C. F. & Chao, Y. C.(1999). Persistent Hz-1 virus infection in insect cells: evidence for insertion of viral DNA into host chromosomes and viral infection in a latent status. Journal of Virology 73, 128-139.
Lossky, M. & Wensink, P. C.(1995). Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor. Molecular and Cellular Biology 15, 6943-6952.[Abstract]
McIntosh, A. H. & Ignoffo, C. M.(1981). Establishment of a persistent baculovirus infection in a lepidopteran cell line. Journal of Invertebrate Pathology 38, 395-403.
Margalit, Y., Yarus, S., Shapira, E., Gruenbaum, Y. & Fainsod, A.(1993). Isolation and characterization of target sequences of the chicken CdxA homeobox gene. Nucleic Acids Research 21, 4915-4922.[Abstract]
Morano, K. A. & Thiele, D. J.(1999). Heat shock factor function and regulation in response to cellular stress, growth, and differentiation signals. Gene Expression 7, 271-282.[Medline]
Nissen, M. S. & Friesen, P. D.(1989). Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. Journal of Virology 63, 493-503.[Medline]
Ogbourne, S. & Antalis, T. M.(1999). Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochemistry Journal 331, 1-14.
Orkin, S.(1992). GATA-binding transcription factors in hematopoietic cells. Blood 80, 575-581.[Medline]
Pullen, S. S. & Friesen, P. D.(1995). The CAGT motif functions as an initiator element during early transcription of the baculovirus transregulator ie-1. Journal of Virology 69, 3575-3583.[Abstract]
Raich, N., Clegg, C. H., Grofti, J., Romeo, P. H. & Stamatoyannopoulos, G.(1995). GATA-1 and YY1 are developmental repressors of the human -globin gene. EMBO Journal 14, 801-809.[Abstract]
Ralston, A. L., Huang, Y. S. & Kawanish, C. Y.(1981). Cell culture studies with the IMC-Hz-1 nonoccluded virus. Virology 115, 33-44.
Ramain, P., Heitzler, P., Haenlin, M. & Simpson, P.(1993). pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1. Development 119, 1277-1291.
Sistonen, L., Sarge, K. D. & Morimoto, R. I.(1994). Human heat shock factors 1 and 2 are differentially activated and can synergistically induce hsp 70 gene transcription. Molecular and Cellular Biology 14, 2087-2099.[Abstract]
Turgores, A., Magness, S. T. & Brenner, D. A.(1994). A single promoter directs both housekeeping and erythroid preferential expression of the human ferrochelatase gene. Journal of Biological Chemistry 269, 30789-30797.
Vaughn, J. L., Goodwin, R. H., Tompkins, G. J. & McCawley, P.(1977). The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213-217.[Medline]
Volkman, L. E., Blissard, G. W., Friesen, P. D., Keddie, B. A., Possee, R. D. & Theilmann, D. A.(1995). Family Baculoviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses , pp. 104-113. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Javis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York:Springer-Verlag.
Yang, H. Y. & Evans, M. T.(1995). Homotypic interactions of chicken GATA-1 can mediate transcriptional activation. Molecular and Cellular Biology 15, 1353-1363.[Abstract]
Received 21 July 2000;
accepted 20 October 2000.