pp38 - Springer Link

Science in China: Series C Life Sciences 2006 Vol.49 No.1 53—62

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DOI: 10.1007/s11427-004-0119-y

The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek’s disease virus DING Jiabo1, CUI Zhizhong1, JIANG Shijin1 & REDDY Sanjay 2 1. Animal Science and Technology College, Shandong Agricultural University, Taian 271018, China; 2. Texas A&M University, College Station, TX 77843, USA Correspondence should be addressed to Cui Zhizhong (email: [email protected])

Received June 19, 2005; accepted September 11, 2005

Abstract There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene and 1.8-kb mRNA transcript gene family in the genome of Marek’s disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP and pP(1.8kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF) and pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF, and no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to cotransfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24 and EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38 and pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction. Keywords: Marek’s disease virus (MDV), pp38 gene, 1.8-kb mRNA transcript, bi-directional promoter, trans-acting factor.

Marek’s disease viruses (MDV) belong to a subgroup of alphaherpesvirus in herpesviridae, and could be divided into 3 serogroups. Among them, serotype 1 www.scichina.com

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could cause lymphoproliferative disease in chickens characterized by the formation of T-cell lymphomas in various visceral organs and tissues.

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Based on molecular virology studies, 4 genes of MDV1 have been shown to be related to the tumorogenecity of MDV: the 1.8-kb mRNA transcript with 132-bp repeats[1,2], the 38kd phosphorylated protein gene (pp38)[3], the meq gene[4], and ICP4[5]. The pp38 is a serotype 1 MDV specific protein, and there is no homolog of pp38 detected in other heresviruses of mammals and the human. The relationship between tumorigenesis and pp38 was first speculated because it was the only MDV-specific antigen detected in all non-producer MD cell lines in the mid 1980s[6,7]. Since the pp38 gene was identified[3], however, little progress has been made concerning the role of pp38 in MDV-transformation. The direct role of pp38 in transformation by MDV was doubted since the pp38 gene is also expressed by the non-oncogenic MDV1 vaccine strain CVI988/Rispens[5,8,9]. Other reports indicated possible roles of pp38 in immunosuppression ― induced by MDV[9 12], and it can be detected in the early stage in MDV’s infection[13]. Recently, by inoculation of MDV-susceptible birds with the pp38 deletion mutant virus, it was reported that pp38 was involved in the early cytolytic infection in lymphocytes but not in the induction of tumor[14]. However the molecular basis for neoplastic transformation of lymphocytes by MDV needs to be further elucidated. Complete 1.8-kb mRNA transcripts are present in oncogenic viruses but are truncated in attenuated variants[15,16], and multiple copies of the 132-bp repeats were found in vaccine strain CVI988 or attenuated viruses compared to the virulent oncogenic strains[17,18]. Interestingly, a short fragment between pp38 gene and 1.8-kb mRNA family on the MDV genome contains a bi-directional transcriptional promoter sequence which controls the transcription of both genes in opposite orientations. Although the promoter sequence is only 305 bp in size, it contains the replication origin and several cis-acting motifs such as TATA-box, CAAT-box, Oct-1, and Sp1[1,3,19]. In the middle of this promoter region, there is a 90-bp putative replication origin of MDV genome[1,20] which shares more than 80% nucleotide identity among three serotypes of MDV, and over 70% identity with those of other α-herpesviruses[21]. When the bi-directional promoter was inserted into plasmids, however, it was found that chloramphenicol acetyltransferase (CAT)

Science in China: Series C Life Sciences

reporter gene under the control of the promoter was expressed transiently only in MDV-infected chicken embryo fibroblasts (CEF) but not in normal CEF, speculating there was a viral or cellular factor(s) involved[22]. In this paper, we report that pp38 itself is one of the viral factors which influence the activity of the bi-directional promoter by comparisons of activities of the promoter for green fluorescence protein gene as a reporter in a pair of molecular clones of MDV, rMd5 and its pp38-deleted mutant rMd5Δpp38. 1 1.1

Materials and methods Materials and reagents

pUC18 vector, T4 ligase, X-Gal, IPTG, and all the enzymes were purchased from Takara Biotechnology (Dalian) Co., Ltd. FITC labeled anti-mouse IgG was purchased from Sigma; LipoFectamine was from GiBco BRL; plasmid purification Mini Kit was from Qiagen; pEGFP-C1 vector was from Clontech; pcDNA3.1/Zeo(+) was from Invitrogen; SPF chicken embryos were from SFAFAS Company (Jinan). The mouse serum anti-pp38 was kindly offered by Dr. Cui Xiaoping. 1.2 Replication of MDV in CEF and extraction of genomic DNA of MDV-CEF In this stuty, several MDV strains were used: virulent MDV (vMDV) strain GA[23], and a pair of molecularly cloned recombinant MDVs, rMd5 and rMd5Δpp38. A pair of cloned viruses were prepared from the original Md5 strain. The difference between them was that the pp38 gene was present in rMd5 but deleted in rMd5Δpp38[14]. All 3 viruses were provided by Avian Disease and Oncology Lab, USDA. Primary CEF was prepared for replication from SPF chicken embryos incubated for 9―11 d. The primary CEF monolayers were inoculated with MDV-infected CEF stocks at a ratio 10:1 of fresh prepared CEF to MDVCEF stock. When plaques induced by MDV-infection were clearly formed, the MDV-CEF monolayers were trypsinized and then spun down. The cell pellets were used for extraction of total genomic DNA by proteinase K and phenol solutions as usual.

Enhancement effect of pp38 gene product on activity of its upstream bi-directional promoter in Marek’s disease virus

1.3

Construction of different recombinant plasmids

(i) Construction of EGFP-expressing plasmids under the control of the bi-directional promoter. For the recombinant plasmids, the bi-directional promoter sequences were amplified by PCR with primers: 5′-GCGAGAGCTCAGAGAGCATCGCGAAGAG-3′ as the forward primer (from base −693 to −676 of pp38 gene[3] plus a Sac I sequence), and 5′-CCTGAGTCGACTTATCCTATACCG-3′ as the reverse primer (from base −325 to −337 plus a Sal I sequence) for the promoter P(pp38) in pp38 transcriptional direction, or with primer: 5′-CCTGAGTCGACTTATCCTATACCG-3′ as the forward primer (from bases −325 to –337 plus a SalI sequence) and 5′-GCGGAGCTCAGAGAGCATCGCGAAGAG-3′ as the reverse primer (from base −693 to −676 plus a Sac I sequence) for the promoter P (1.8-kb) in the 1.8-kb mRNA transcript direction. The putative PCR product of 369 bp contained the whole promoter-enhancer of 305 bp[22,24]. EGFP gene was amplified by PCR from pEGFP-C1 (Clontech, PT3028-5, the PCR product of EGFP is relative to bases 606―1330 of the plasmid) and inserted into pUC18 at the sites of Sal I and Sph I. Then, the bi-directional promoters P (pp38 ) and P (1.8-kb) PCR products prepared as above were inserted at Sac I and Sal I sites to obtain recombinant plasmids pP(pp38)EGFP and pP(1.8-kb)-EGFP expressing EGFP under regulation of promoters in opposite directions. (ii) Construction of pp38- and pp24-expressing plasmids. To construct the recombinant plasmid pcDNA-pp38, pp38 gene (1―873 bp) was amplified with primers: 5′-AATGGATCCACTCATGACCCACATGGAA-3′ as forward primer (from bases −13 to 6, plus a BamH I linker), and 5′-CGAGGTCGACATCGGGTACGGCTACACTG-3′ as the reverse primer (from bases 882 to 900, plus a Sal I linker). PCR product was cloned into pcDNA-3.1/Zeo(+) vector (Invitrogen) by the enzyme BamH I and Sal I. To construct the recombinant plasmid pcDNA-pp24, the whole pp24 gene was amplified with primers according to the sequence published and inserted into pcDNA-3.1/Zeo(+) vector at sites of BamH I and Sal I. All the plasmids were purified with plasmid purifi-

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cation. Mini Kit was from Qiagen and quantified by comparisons with standard DNA markers in gel electrophoresis before transfection. 1.4 Transfection of different MDV-CEF with plasmids pP(pp38)-EGFP and pP(1.8-kb)-EGFP (i) Preparation of CEF or MDV-CEF cell monolayer for transfection. To prepare the secondary CEF monolayers, 1×106 cells were inoculated into 35 mm dishes with 2 pieces of coverslips for transfection, and incubated at 37℃ for 18―24 h for cell monolayers to be formed. To prepare the secondary MDV-infected CEF monolayers, primary CEF monolayers were prepared in a 60 cm2 flask and inoculated with rMd5- or rMd5Δpp38-CEF stocks of about 1×105 plaque form unit (pfu). They were incubated for 3―4 d until cell pathogenic effect was demonstrated in about a quarter of cells in the monolayers. The MDV-CEF monolayers were resuspended by treatment of trypsin solution. One part of the MDV-CEF suspension was mixed with two parts (by cell number) of fresh secondary CEF suspension and placed into 35 mm dishes (1×106 cells per dish). Transfection was carried out when the monolayers formed about 18 h later. (ii) Transfecion of CEF or MDV-CEF with DNA of different plasmids. Transfection of recombinant plasmid DNA was performed by using LipofectAMINETM reagent (Gibco BRL) according to the manufacturer’s instructions. Briefly, 2 μg plasmid DNA and 4 μL LipofectAMINETM reagent (Gibco BRL) were added into two polypropylene tubes with 100 μL of DMEM medium free of serum and antibiotic. Two solutions were mixed and incubated for 45 min at room temperature and then added into another 800 μL DMEM. Totally 1 mL of transfection solution was carefully poured onto the cell monolayers in 35 mm dishes with cell monolayers. Eight hours later, 1 mL of complete medium with 10% bovine fetus serum was added to the transfected cell monolayers. All dishes were maintained at 37℃ in a CO2 incubator. The expression of EGFP was determined 24 h and 48 h post transfection. When pcDNA-pp38 was co-transfected with pP(pp38)-EGFP or pP(1.8-kb)-EGFP, 2 μg plasmid DNA each was mixed with 4 μL LipofectAMINETM reagent for transfection in a 35 mm dish.

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1.5


Detection of EGFP in transfected CEF

24 h and 48 h post transfection, the cell monolayers on coverslips were directly examined under a fluorescence microscope (Nikon Eclipse E600) for fluorescence from EGFP expressed in tranfected CEF cells. 1.6 Detection of pp38 in transfected CEF with indirect fluorescence antibody test (IFA) IFA with anti-pp38 mouse serum (mice were immunized with pp38 expressed in E. coli, offered kindly by Dr. Cui Xiaoping) was used to detect the expression of pp38 in CEF transfected with pcDNA-pp38 plasmid DNA or rMd5Δpp38-infected CEF monolayers on coverslips picked up 24 and 48 h after transfection. For IFA, the cell monolayers on the coverslips were fixed with cold acetone:alcohol mixture (2:1), and then 50 μL of 1:500 diluted anti-pp38 mouse serum was added and incubated for 45 min at 37℃. After washing 3 times with 1× PBS (pH=7.2), 50 μL of 1:256 diluted anti-mouse IgG sheep serum conjugated by FITC (Sigma) was added and incubated for 1 h at 37℃. After washing 3 times, the coverslips were observed under a fluorescence microscope (Nikon Eclipse E600). Un-immunized mouse serum was used as a control. 1.7 Co-transfection of uninfected CEF cells with pcDNA-pp38, pcDNA-p24 and EGFP-expressing recombinant plasmids CEF monolayers prepared on coverslips in 35mm dishes were transfected with pcDNA-pp38, pcDNAp24 and pP(pp38)-EGFP/pP(1.8-kb)-EGFP 2 μg each in different combinations. 48 h post transfection, EGFP was detected under UV light with a fluorescence microscope. 1.8

Mobility shift DNA-binding assay

(i) Preparation of cytoplasmic extracts for DNAbinding assay. CEF monolayers in 60 mm dishes were transfected with plasmids pcDNA-pp38 or pcDNA-pp24 alone or co-transfected with both. 48 h after transfection, the culture medium was removed and cell monolayers were washed with 1×PBS 3 times. Then the transfected cells were scraped into fresh 1× PBS in a 1.5 mL tube and centrifuged at 1850 g for 5 min. The packaged cell pellets were re-suspended into

5 times (v/v) of hypotonic buffer (10 mmol/L HEPES (pH=7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2 mmol/L PMSF, 0.5 mmol/L DTT) and centrifuged for another 5 min at 1850 g. They were re-suspended in 3 times (v/v) of the hypotonic buffer as the original packed cell pellet and cells were swelled on ice for 10 min or even longer till more than 80% of the cells were lyzed. Next, the lysate was centrifuged for 15 min at 3300 g and the supernatant was collected. After adding 0.11 volume of 10× cytoplasmic buffer (0.3 mol/L HEPES (pH=7.9), 1.4 mol/L KCl, 0.03 mol/L MgCl2) in the saved supernatant, they were centrifuged for 1 h at 100000 g. Supernants were dialyzed against 50 volumes of dialysis buffer (20 mmol/L HEPES, 20% (v/v) glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 0.2 mmol/L PMSF, 0.5 mmol/L DTT) for 2 h, and then removed to a 1.5 mL centrifuge tube for centrifugation at 25000 g for 20 min. The supernatants were aliquoted into sample tubes of 40 μL each and stored at −80℃ for use. (ii) Preparation of Dig-labeled DNA probes of the promoter DNA fragments for mobility shift assay. Three single stranded DNA fragments representing 3 connective subregions I, II and III of the bi-directional promoter (Fig. 1) were synthesized by commercial service (Bioasia company, Shanghai). They were 67, 73 and 58 pb long respectively. At the same time, 3 primers of 10 bp complementary to 3′-end of subregions I, II and III were also synthesized. The dig-labeled double stranded DNA probes corresponding to subregions I, II and III were prepared by DIG DNA Labeling and detection Kit (Roche, Cat. No. 1093657) as follows: The 3 single stranded DNA of subregions were used as templates, and their complementary strands were synthesized with their own 10 bp primers complementary to 3′-end of each subregion by klenow enzyme in dNTP and Dig-dTTP labeling mixture. The detail is according to the manufacturer’s instruction. The final dig-labeled DNA probe solutions of 20 μL were from labeling reactions containing 1 μg of originally synthesized single stranded DNA as templates. (iii) Mobility shift DNA binding and detection. In a microcentrifuge tube, 40 μL cytoplasmic extract from CEF transfected with different plasmids was in-


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Fig. 1. Demonstration of the structure of the bi-directional promoter and locations of the 3 subregions used for DIG labeling DNA as probes in the promoter. The sequence number was according to the previous publication (Cui et al. [3]).

cubated with the following 20 μL probe working solutions: 2 μL digoxigenin-labeled DNA probes (relative to subregion I, II or III), 18 μL of 300 μg/mL BSA in distilled water, kept at 30℃ for 15 min. Afterwards, the samples were applied to a nondenaturing 4% polyacrylamide gel for electrophoresis. Then the gel was transferred to a piece of nitrocellulose membrane (NC). The free probe or protein-bound probe DNA on the NC was detected in further immunological reactions by adding anti-Digoxigenin-AP conjugate and then NBT/BCIP solution according to the manufacturer’s instruction of the Digoxingenin-labeling and detection Kit (Roche, Cat. No. 1093657).

with typical MDV plaques in rMd5-infected CEF monolayer. However, no green fluorescence could be detected in the CEF surrounding the MDV plaques in rMd5Δpp38-CEF monolayer 2―4 d after transfection with pP(pp38)-EGFP or pP(1.8-kb)–EGFP (Fig. 2(b) and (e)). These results suggest that the pp38 gene product may play an important role for the activity of the bi-directional promoter to transcribe the EGFP gene. The fluorescent intensity in rMd5-CEF transfected with pP(1.8-kb)–EGFP was much higher than that with pP(pp38)-EGFP (Table 1). It implies that the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene.

2

2.1 Expression of EGFP in different cells transfected with different EGFP reporter plasmids alone

2.2 The expression of EGFP in different cells co-transfected with pcDNA-pp38 and EGFP reporter plasmids

To analyze the regulation activity of the bi-directional promoter for EGFP reporter gene expression, pP(pp38)-EGFP and pP(1.8-kb)- EGFP were used to transfect CEF monolayers infected with rMd5, rMd5Δpp38 or uninfected CEF. The result showed that EGFP expression could be detected only in rMd5CEF transfected with both pP(pp38)-EGFP and pP(1.8-kb)EGFP (Table 1 and Fig. 2(a) and (d)), but not in uninfected CEF or rMd5Δpp38-CEF. Under a fluorescence microscope as indicated in Fig. 2(d), the green fluorescence from EGFP was directly demonstrated in the spindle-shape CEF surrounding or mixed

In IFA, pp38 was easily detected with anti-pp38 mouse serum in the cytoplasm of uninfected CEF transfected with plasmid pcDNA-pp38 24 h post transfection (Fig. 3). All transfected CEF were negative in IFA with un-immunized mouse serum, untransfected CEF and rMd5Δpp38-CEF were also negative in the same IFA with the anti-pp38 serum (data not included). The fluorescence of EGFP was detected in rMd5Δpp38-CEF co-transfected with EGFP reporter and pcDNA-pp38 (Fig. 2(f) and Table 1) when no EGFP expression was demonstrated in rMd5Δpp38-

Results

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Fig. 2. Photos taken under regular light (capital letter) or UV light (small letter) from the same plaques in the same visual fields of different transfected or co-transfected MDV-CEF with different plasmids (×200). (a) and (d), The rMd5-CEF transfected with pP(1.8kb)-EGFP only; (b) and (e), the rMd5Δpp38-CEF transfected with pP(1.8kb)-EGFP; only (c) and (f), the rMd5Δpp38-CEF cotransfected with plasmids pP(1.8kb)-EGFP and pcDNA-pp38. Table 1 Comparison of fluorescence intensity from EGFP expressed under the control of bi-directional promoters in different MDV-CEF or normal CEF transfected with different plasmid or their combinations Transfected with plasmidsa) CEF

pUC-EGFP

pP(pp38)-EGFP

pP(1.8-kb)-EGFP

pUC-EGFP＋ pcDNA-pp38

pP(pp38)-EGFP＋

pP(1.8-kb)-EGFP＋

pcDNA-pp38

pcDNA-pp38

Uninfected

−

−

−

−

−

−

rMd5∆pp38

−

−

−

−

+

++

rMd5

−

++

++++

ND

ND

ND

a) “–”, “+”, “++”, “++++” represent the expression levels of EGFP according to the fluorescence intensity judged under an UV microscope.

Fig. 3. Detection of pp38 by IFA with anti-pp38 mouse serum in CEF transfected with reporter plasmid pcDNA-pp38 (×200). The fluorescence was very clear in cytoplasm.

Fig. 4. The fluorescence on CEF cells co-transfected by pcDNA-pp38, pcDNA-pp24 and EGFP reporter plasmid (×200). (a) Co-transfected with pcDNA-pp38, pcDNA-pp24 and pP(pp38)-EGF; (b) co-transfected with pcDNA-pp38, pcDNA-pp24 and pP(1.8-kb)-EGFP.

CEF transfected with EGFP reporter plasmids only (Fig. 2 (e)). The result confirmed that pp38 is necessary for the activity of the promoter. In addition to pp38, other factor(s) from MDV-infection were also

involved in the activity of the promoter, since no EGFP activity could be detected in uninfected CEF even co-transfected with pcDNA-pp38 and pP(1.8kb)-EGFP or pP(pp38)-EGFP (Table 1).


2.3 Comparison of the activities of the bi-directional promoters in two opposite directions In the transfected CEF, the ratio of positive cells and intensity of fluorescence from plasmid pP(1.8-kb)EGFP was higher than that from plasmid pP(pp38)EGFP (Table 1), indicating that the activity of the bi-directional promoter for 1.8-kb mRNA direction was stronger than that for the pp38 direction.

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of 67 bp from base −581 to −515 or 57 bp from base −441 to −384 ) were not influenced by the same cytoplasmic extracts in their mobility (data not shown). The results indicated that the fragment of 73 bp from base −514 to −442 in the bi-directional promoter could be bound by pp38 when pp24 was also co-expressed.

2.4 The expression of EGFP in uninfected CEF cells co-transfected with pcDNA-pp38, pcDNA-pp24, and EGFP reporter plasmids 48 h post co-transfection with pcDNA-pp38 and pcDNA-pp24, and EGFP reporter plasmids, EGFPfluorescence was detected in 2% of cells co-transfected with pP(pp38)-EGFP and in 5% of cells cotransfected with pP(1.8-kb)-EGFP (Fig. 4). But no EGFP activity could be detected when either pcDNApp38 or pcDNA-pp24 alone was used in co-transfection with EGFP reporter plasmids. It suggested that pp24 was another factor involved in regulation of the promoter. pp24 and pp38 worked together to make the promoter effective in CEF. And the fluorescence in pP(1.8-kb)-EGFP transfected cells was stronger than that in pP(pp38)-EGFP transfected cells. Fig. 4 also demonstrates that the activity of the bi-directional promoter for 1.8-kb mRNA direction was stronger than that for the pp38 direction as in Table 1 for rMd5∆pp38-CEF. 2.5 Determination of pp38-bound fragments of the bi-directional promoter by DNA mobility shift assays Digoxigenin-labeled DNA fragments relative to 3 subregions (Fig. 1) of the promoter were used in DNA mobility shift assays when they were treated with cellular extracts of CEF transfected with recombinant pcDNA-pp38, pcDNA-pp24 or both. Only the subregion Ⅱ of 73 bp from base −514 to −442 relative to pp38 ORF (Fig. 1) was retarded in gel electrophoresis when treated with cellular proteins extracted from CEF co-transfected with both pcDNA-pp38 and pcDNA-pp24 (Fig. 5). The cellular extracts from CEF transfected with pcDNA-pp38 or pcDNA-pp24 alone or from un-transfected CEF did not show any effect on the mobility of the same DNA fragment probe. Other two subregion DNA fragments ( relative to sequences

Fig. 5. Gel mobility shift assay of Dig-labeled subregion II DNA fragment of 73 bp. Lane 1, Treated with the cellular extract of normal CEF (negative control); lane 2, treated with the cellular extract of CEF co-transfected with both pcDNA-pp38 and pcDNA-pp24; lane 3, treated with the cellular extract of CEF transfected with pcDNA-pp38 alone; lane 4, treated with the cellular extract of CEF transfected with pcDNA-pp24 alone; lane 5, no treatment with any cellular extract. After Dig-labeled DNA probes were transferred onto the nitrocellulose membrane from gel, an immune reaction was conducted with alkaline phosphatase-conjugated anti-dig antibody. DNA bands were demonstrated in color in NBT/BCIP solution. The retarded band was demonstrated when compared with the original Dig-labeled subregion II fragment of 73 bp in all 5 lanes.

3

Discussion

The pp38 gene is a unique gene which has been detected only in MDVs so far. No homologues have been found in other groups of herpesviruses or in any other genomes since the gene was identified and sequenced[3,26]. Its biological functions have been studied from different aspects by various methods. As an immediate early gene, pp38 was the MDV-specific antigen detected more early and in higher percentages of MDV-infected cells or MDV-transformed cell line cells than other antigens, such as meq and gB[13], it was actively expressed in lytically infected cells[7]. So far several different possible biological roles of pp38 were speculated, such as maintenance of the transformation of MDV cell line proliferation[5] and immunosuppression induced in MDV infection. For example, our repeated experiments indicated that the recombinant pp38 expressed in insect Sf9 cells using a bacu-

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lovirus vector had immunosuppressive effects in chickens to certain antigen such as mouse red blood cells[10]. It was reported that high levels of expression of MDV pp38 considerably reduced MHC-restricted cytolysis by splenocytes from chickens 7 d post inoculation with reticuloendotheliosis virus (REV)[9,12]. When the differential susceptibility to MDV between the susceptible chicken line 72 and another resistant line 61 was studied, it was found that susceptibility was associated with greater numbers of pp38+ cells[11]. Recently, it was also demonstrated that pp38 was involved in early cytolytic infection in lymphocytes but not in the induction of tumors since inoculation of MDV-susceptible birds with the pp38 deletion mutant virus was still able to induce tumors[14]. It has been recognized for many years that there was a bi-directional promoter of about 300 bp between the transcription start sites of the pp38 gene and 1.8-kb mRNA transcript[1,3,20]. The promoter contained several enhancer motifs including the Sp1, Oct1, CAAT and two TATA boxes. In addition, a DNA replication origin and 17 bp-reverse repeats were located within the promoter. Recently, we demonstrated that the pp38 gene could be expressed under the control of the bi-directional promoter in transfected CEF with recombinant plasmid DNA[24,27]. However, it was reported that CAT activity under the control of the bidirectional promoter was only detected in MDV-infected CEF but not in uninfected CEF when transfected with CAT reporter plasmids, indicating that the bi-directional promoter needs to be regulated by the viral or cellular factor(s) induced by viral infection[22]. In this study, we are the first to report that pp38 itself could act as a part of trans-acting factor(s) influencing the activity of the bi-directional promoter. The pair of infectious clones of the Md5 strain, rMd5 and rMd5Δpp38 used in this study, were very helpful, leading us to understand some biological functions of pp38. By analyzing the expression activity of EGFP in rMd5-CEF or rMd5Δpp38-CEF and uninfected CEF transfected with two EGFP reporter plasmids, the effect of pp38 on the bi-directional promoter was obvious. EGFP could be expressed in rMd5-CEF, but not in rMd5Δpp38-CEF. When pP(pp38)-EGFP or pP(1.8-kb)-EGFP was co-transfected with pcDNA-pp38, EGFP could be detected in


rMd5Δpp38-CEF. This indicates that pp38 was essential for the activity of the bi-directional promoter in two orientations as Meq could transactivate its own promoter[28]. While the same co-transfection of CEF did not induce the expression of EGFP. There may be some other factor(s) involved in stimulating the activity of the promoter besides pp38. Before this study, it had been reported that the bi-directional promoter activity in two opposite orientations was regulated by common promoter-specific enhancers with a viral or cellular factor(s) induced by MDV infection. Such factor(s) could bind to a 30 bp fragment in the promoter region[22]. To further prove whether pp38 was able to be a trans-acting transcriptional factor, the DNA mobility shift assay was conducted. In the preliminary test, however, pp38 did not show any expected influence on the mobility of the dig-labeled promoter fragment probes (data not included). In immunoprecipitation of the early report, a monoclonal antibody (mAb) H19 could precipitate both pp38 and pp24[29]. Although there were identical N-terminals of 95 amino acids between pp38 and pp24[30], pp24 did not have the H19-epitope, which was closely related to the 107 amino acid on pp38[31,32]. In Western blot of MDV-CEF lysates, mAb H19 recognized only pp38 but not pp24 (our unpublished data). These results implied that pp24 was co-immunoprecipitated with pp38 by mAb H19 in immunoprecipitation, and pp38 might form a hetero-dimer with pp24. More mobility shift assays convincingly proved that cytoplasmic extracts containing co-expressed pp38 and pp24 could specifically bind to the dig-labeled DNA probe (Fig. 5) corresponding to subregion II in Fig. 1, when the cellular extracts with pp38 or pp24 alone showed no such effect. The result of DNA mobility shift assays was coincident with the EGFP-expression tests, i.e. the co-transfection of pcDNA-pp38 and EGFP reporter plasmids demonstrated the expression of EGFP when transfected into rMd5Δpp38-CEF, but not expressed in uninfected CEF (Table 1). The results indicated that pp38 itself was only a part of the complete trans-acting factor(s). Another protein may work together with pp38 to optimize its role as a trans-acting factor. Since the subregion II bound by pp38/pp24 in this study was 73 bp and covered the 30 bp fragment reported by Shigekane


et al.[22], it is not clear if the pp38/pp24 bound the same sequence or not. The exact sequence bound with pp38/pp24 was to be further identified. Besides, it is noticed that a 7 bp sequence TTCGCAC as a motif bound by origin-binding protein (OBP)[32] was also located in the subregion Ⅱ(Fig. 1), and it overlapped the 30 bp sequence above. OBP had a size of 95 kDa and was apparently different from pp38/pp24. It was reported that CAT-activity expressed under the promoter in the direction for 1.8-kb transcript was always higher than that in the pp38 direction[22]. The present study confirmed their result in transfected rMd5-CEF and rMd5Δpp38-CEF. From these studies, we suggested that pp38, with the help of pp24, upregulates the transcriptional level of itself and 1.8-kb mRNA transcript as meq[28]. Ackoowledgements The authors thank Dr. Cui Xiaoping of Michigan State University for pp38, specific anti-serum. This work was supported by the National Natural Science Foundation of China (Grant Nos. 30300450 & 30070544).

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