The Molecular Characteristics Of Pp38 Gene And The Bi-directional Promoter On The Upstream Of Pp38 Gene Of Marek's Disease Virus

Marek's disease virus (MDV) is a kind of high contagious and tumourgenic herpesvirus to susceptible chicken flocks. Its 38-kDa phosphorylated protein (pp38) is a unique gene in MDV, no homologues have been found in other groups of herpesviruses even in any other organisms. Several possible biological roles of pp38 were speculated, such as maintenance of the transformation of MDV cell line proliferation, MDV infection-induced immunosuppression and cytolytic infection of B-lymphocytes, but little progress has been reported concerning the molecular function of pp38. Between genes pp38 and 1.8-kb mRNA transcript family, there was a nearly 300-bp bi-directional promoter, which contains several enhancer motifs and MDV replication origin. It was reported that some unknown factors associated with the infection of MDV could regulate the activity of this bi-directional promoter. Based on above backgrounds, we carried out a series studies on the structure and activity of the promoter, and the interactions between the pp38 and the promoter. From this study, some more biological functions of pp38 were reported as followings: 1. Comparisons of the promoter sequences in MDV strains of different pathotypes. Polymerase chain reaction (PCR) was used to amplify the promoter sequence from genomic DNA of 9 MDV strains. These viruses were: vaccine strains CVI988 and 814, virulent reference strain GA, very virulent reference strains RB1B, Md5 and Md11,very virulent plus reference strain 648A,and two Chinese field strains GD2 and J-E. Sequence analysis showed that the bi-directional promoters among different MDV strains were highly conserved with a homology of over 95.9% and most mutations occurred near the replication origin. On the phylogenetic tree, two vaccine strains CVI988 and 814 were more closely than others. However, no corelations between the promoter sequences and pathotypes in other virulent strains were found. The promoter of CVI988 strain had a 5-bp deletion, which resulted in a sp1 site deletion, and it was able to be distinguished from other MDV strains by PCR using a pair of primers designed according to the deletion site. 2. Demonstration of the activity of the promoter in two orientations by transfection of cells with pp38 reporter plasmids. To characterize the promoter activity in vitro, the pp38 gene was cloned into pUC18 for plasmid pUC18-pp38, then a 789-bp fragment containing the bi-directional promoter was amplified from the genome of MDV GA strain and inserted at the upstream of pp38 gene in two different directions. Two recombinant pp38-expressing plasmids, pP(pp38)-pp38 (with the promoter in direction for pp38) and pP(1.8kb)-pp38 (with the promoter in direction for 1.8-kb mRNA), were constructed. Chicken embryo fibroblast (CEF) cells were transfected with these two recombinant plasmids, respectively. The expression of pp38 was detected by immunofluorecent assay (IFA) using mouse anti-pp38 serum. Twenty-four hours post transfection, the pp38 protein was detected in CEFs transfected with each of the two recombinants. Forty-eight hours after transfection, 40% of the transfected cells were IFA-positive. This result showed that the bi-directional promoter has the transcriptional activity in both orientations in vitro. 3. Determination of the minimal sequence of the promoter for the full transcriptional activity with pp38 reporter plasmids. By using a PCR-based deletion strategy, different subfragments of the 789-bp promoter sequence were prepared and cloned into pUC18-pp38 at the upstream of pp38 gene. Then six plasmids, including pP(pp38)-pp38-1,pP(pp38)-pp38-2,pP(pp38)-pp38-3, pP(1.8kb)-pp38-1,pP(1.8kb)-pp38-2 and pP(1.8kb)-pp38-3 were constructed. The above 6 constructs and plasmids pP(pp38)-pp38, pP(1.8kb)-pp38 were transfected into CEF, respectively. Forty-eight hours post transfection, IFA was applied to detect the expression of pp38 by the fluorescent density in IFA. It was determined that a minimal sequence of 320-bp as the core region was necessary for the full transcriptional activity of the promoter. 4. Testifying the activity of the two single-direction promoters divided from the bi-directional promoter, and confirming that the full transcript activity depends on the intact structure of the bi-directional promoter. On each side of the replication origin within the bi-directional promoter, there were claimed TATA-box and CAAT-box. To investigate whether the bi-directional promoter contains two independent promoters, the bi-directional promoter was cut at the replication origin region to give two fragments, which were subcloned into pUC-pp38 vector, respectively. The resulting vectors pdP(pp38)-pp38 and pdP(1.8kb)-pp38 were used to transfect MDV-infected CEF cells. Twenty-four hours after transfection, the expression of pp38 was detected by IFA. It seemed like that these two subunits of the promoter have transcriptional activity independently. To analyze the promoteractivity quantitatively, the intact promoters and its subunits were cloned into pCAT-Basic vector at the upstream of chloramphenicol acetyltransferase (CAT) gene, respectively. The resulted four plasmids pP(pp38)-CAT, pP(1.8kb)-CAT, pdP(pp38)-CAT and pdP(1.8kb)-CAT were used to transfect MDV GA infected CEF. Forty-eight hours post transfection, CAT activities were detected in the lysates of the samples transfected by plasmids. Under the control of two subunits of the bi-directional promoter, CAT activitis were much lower than that under the control of the intact promoters. Especially for the 1.8-kb mRNA direction, for example, CAT activity in cells transfected with pdP(1.8kb)-CAT (only contained a subunit of the intact promoter) was only 1/41 as that in cells transfected with pP(1.8kb)-CAT (with intact promoter). These results indicated that the bi-directional promoter is not simply a tandem repeats of the two subunits, its full activity depended on its complete structure. 5. The pp38 is one of factors up-regulating the activity of the promoter. The above experiments indicate that the bi-directional promoter can drive transcription of the pp38 gene in CEF cells without MDV infection, whereas MDV infection is essential for the expression of CAT reporter gene, further indicating that pp38 is the transactivator of the bi-directional promoter. To verify this hypothesis, enhanced green fluorescent protein (EGFP) gene was cloned into vectors under the control of the bi-directional promoter in two opposite directions. Both EGFP-expressing plasmids (pP(pp38)-EGFP and pP(1.8kb)-EGFP) and CAT-expressing plasmds (pP(pp38)-CAT and pP(1.8kb)-CAT) were transfected to CEF infected with MDV clone rMd5 (rMd5-CEF) or pp38-deleted rMd5?pp38 (rMd5?pp38-CEF), respectively. As the results, EGFP was expressed only in rMd5-CEF, but not in uninfected CEF or rMd5?pp38-CEF. CAT was expressed in rMd5?pp38-infected CEF, but the activity was 3.5-fold (pP(pp38)-CAT ) and 15-fold (pP(1.8kb)-CAT) lower than that in rMd5-CEF, demonstrating that pp38 had significant influence on the promoter activity. To testify the influence of pp38 to the promoter, the pp38-expressing plasmid pcDNA-pp38 was constructed for co-transfection with EGFP-or CAT-expressing plasmids. The results showed that EGFP was expressed in rMd5?pp38-CEF co-transfected with pcDNA-pp38 and pP(pp38)-EGFP/pP(1.8kb)-EGFP, but not in uninfected CEF. CAT activity was significantly higher in rMd5?pp38-CEF co-transfected with pcDNA-pp38 and pP(pp38)-CAT/pP(1.8kb)-CAT, but no activity was detected in uninfected CEF, indicating thatother trans-acting factor(s) was required for the activity of the promoter besides pp38. 6. Immunoprecipitation of fluorescence-labeled cell lysates demostrates that pp38 and pp24 could form a heteropolymer. The pp38-expressing plasmid pcDNA-pp38, pp24-expressing plasmid pcDNA-pp24 and pp38 and pp24 co-expressing plasmid pBud-pp38-pp24 were transfected into uninfected CEF, respectively. Forty-eight hours after transfection, the transfected cells were lyzed for immunoprecipitation with pp38-specific moloclonal antibody H19 (Mab H19). The pp24 was immunoprecipitated in the lysate of pBud-pp38-pp24-transfected cells and pcDNA-pp38 and pcDNA-pp24 co-transfected cells, but not in pcDNA-pp38 or pcDNA-pp24 transfected cells. These results indicate that pp38 and pp24 can form a structural heteropolymer. 7. It was the pp38/pp24 heteropolymer that up-regulates the transcriptional activity of the promoter. The above studies proved that pp38/pp24 was a heteropolymer and pp38 expressed alone could not activate the promoter. To testified whether the heteropolymer pp38/pp24 could regulated the promoter, pp38 and pp24 expressing plasmids were co-transfected with pP(1.8kb)-EGFP and pP(1.8kb)-CAT plasmids. When pBud-pp38-pp24 was co-transfected with pP(1.8kb)-EGFP , 48h post transfection, green fluorescence could be detected on uninfected CEF, and the percentage of EGFP-positive cells was about 0.5%. No EGFP-positive cells was detected in CEF co-transfected with pP(1.8kb)-EGFP and pcDNA-pp38 or pcDNA-pp24. In cells co-transfected with plasmids pP(1.8kb)-CAT and pBud-pp38-pp24, high CAT activity was detected 48h post transfection. Similarly, no CAT activity was detected in cells co-transfected with pcDNA-pp38 or pcDNA-pp24. These results confirmed that pp38 and pp24 were in form of a heteropolymer, which can transactivate the bi-directional promoter on the upstream of pp38 gene. 8. DNA mobility shift assay proved that the heteropolymer pp38/pp24 could bind a 73-bp region of the promoter. To investigate whether pp38 could bind the bi-directional promoter, DNA mobility shift assay was conducted, in which 3 single-stranded overlapping DNA fragments of 67-bp (fragmentⅠ), 73-bp (fragment Ⅱ) and 58-bp (fragment Ⅲ) spanning the bi-directional promoter were used. In addition, 3 primers of 10-bp complementary to the 3′-ends of fragment Ⅰ, Ⅱand Ⅲwere synthesized. Three double-stranded fragments were generated from the single-stranded fragments by Klenowenzyme in presence of DIG-labeled dNTPs. Cellular extracts from CEF transfected with pcDNA-pp38 and/or pcDNA-pp24 were incubated with the 3 DIG-labeled fragments, respectively. As the results, only the samples co-expressing pp38 and pp24 could bind the fragment Ⅱ(73-bp,-514～-442, relative to pp38 ORF), and a special retarded band was found in electrophoresis, just as the extract samples of CEF infected by MDV. No retarded band was evident in pcDNA-pp38-or pcDNA-pp24-transfected cells. Western blotting with MDV specific Mab H19 showed that pp38 was the 73-bp fragment binding protein. 9. The 9-bp sequence (5'-CTGCTCATT-3', -461~-469) bound by heteropolymer pp38/pp24 was determined by competitive inhibition assay in DNA mobility shift test. To determine the minimal sequence bound by pp38/pp24, the 73-bp fragment was further divided into three overlapping fragments (fragment 1, 2, 3). The three double-stranded fragments were generated from single-stranded fragments and their complementary fragments by annealing. These three double-stranded fragments were excessively added to the lysate of CEF co-expressing pp38 and pp24, and DIG-labeled 73-bp fragment was added as well as before. Analyzing by DNA mobility shift assay, a 26-bp of fragment 3 (-467～-442) could competitively inhibit the binding of pp38/pp24 to DIG-labeled 73-bp sequence, and made the retarded band disappear. The similar way was used for the division of the 26-bp oligonucleotides. After 4 trials of division, a 9-bp binding sequence of pp38/pp24 (5'-CTGCTCATT-3', -461~-469) was finally determined. 10. One sp1 deletion in the promoter caused 20% of decrease to the transcriptional activity for the direction of 1.8-kb mRNA, in which direction the activity was significantly stronger than that in pp38. To analyze the effect of sp1 deletion on the bi-directional promoter activity, promoters PCVI (pp38) and P CVI (1.8kb) were amplified by PCR from the genomic DNA of MDV CVI988 strain and subcloned into pCAT-basic vector to give plasmids pPCVI(pp38)-CAT and pPCVI(1.8kb)-CAT. pP(pp38)-CAT, pP(1.8kb)-CAT, pP CVI (pp38)-CAT and pPCVI(1.8kb)-CAT were transfected, respectively, to GA infected CEF cells and CAT activities were measured. The results showed that sp1 deletion decreased the promoter activity by 20% for 1.8-kb mRNA transcription, but had little influence on pp38 gene transcription. The CAT activity in the sample transfected by pP(1.8kb)-CAT was 15 times as that in pP(pp38)-CAT sample, which indicates the promoter activity for 1.8-kb...