- Open Access
The construction and characterization of the bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts of Marek's disease viruses
© Chen et al; licensee BioMed Central Ltd. 2009
- Received: 14 October 2009
- Accepted: 30 November 2009
- Published: 30 November 2009
Marek's disease virus (MDV) has a bi-directional promoter between pp38 gene and 1.8-kb mRNA transcripts. By sequencing for the promoters from 8 different strains (CVI988, 814, GA, JM, Md5, G2, RB1B and 648A), it is found, comparing with the other 7 MDV strains, CVI988 has a 5-bp (from -628 to -632) deletion in this region, which caused a Sp1 site destroyed. In order to analysis the activity of the promoter, the complete bi-directional promoters from GA and CVI988 were, respectively, cloned into pCAT-Basic vector in both directions for the recombinants pPGA(pp38)-CAT, pPGA(1.8 kb)-CAT, pPCVI(pp38)-CAT and pPCVI(1.8 kb)-CAT. The complete promoter of GA was divided into two single-direction promoters from the replication of MDV genomic DNA, and cloned into pCAT-Basic for pdPGA(pp38)-CAT and pdPGA(1.8 kb)-CAT as well. The above 6 recombinants were then transfected into chicken embryo fibroblasts (CEFs) infected with MDV, and the activity of chloramphenicol acetyltransferase (CAT) was measured from the lysed CEFs 48 h post transfection.
The results showed the activity of the divided promoters was decreased on both directions. In 1.8-kb mRNA direction, it is nearly down to 2.4% (19/781) of the whole promoter, while it keeps 65% (34/52) activity in pp38 direction. The deletion of Sp1 site in CVI988 causes the 20% activity decreased, and has little influence in pp38 direction.
The present study confirmed their result, and the promoter for the 1.8-kb mRNA transcripts is a much stronger promoter than that in the orientation for pp38.
- Enhance Green Fluorescence Protein
- Chicken Embryo Fibroblast
- Chicken Embryo Fibroblast Cell
- Pp38 Gene
- CVI988 Strain
Marek's disease virus (MDV) is an oncogenic herpesvirus, which causes a highly contagious neoplastic disease in chickens, and could be divided into 3 serogroups. Among them, serotype 1 could cause lymphoproliferative disease in chickens characterized by the formation of T-cell lymphomas in various visceral organs and tissues.
Based on molecular virology studies, 4 genes of MDV1 have been shown to relate to the tumorogenecity of MDV: the 1.8-kb mRNA transcript with 132-bp repeats[2, 3], the 38 KD phosphorylated protein gene (pp38), the meq gene , and ICP4. 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 [7, 8]. Complete 1.8-kb mRNA transcripts are present in oncogenic viruses but are truncated in attenuated variants [9, 10], and multiple copies of the 132-bp repeats are found in vaccine strain CVI988 or attenuated viruses compared to the virulent oncogenic strains [11, 12].
Interestingly, a short fragment between pp38 gene and 1.8-kb mRNA family on the MDV genome contains a bi-directional transcriptional promoter sequence that 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[2, 4, 13].
In the middle of this promoter region, there is a 90-bp putative replication origin of MDV genome [2, 14], which shares more than 80% nucleotide identity among three serotypes of MDV, and over 70% identity with those of other α-herpesviruses . When the bi-directional promoter was inserted into plasmids, however, it was found that chloramphenicol acetyltransferase (CAT) reporter gene under the control of the promoter was expressed transiently only in MDV-infected chicken embryo fibroblasts (CEF) but not in normal CEFs, speculating there was a viral or cellular factor(s) involved . Our previous study showed pp38 could enhance the bi-directional promoter activity between pp38 gene and 1.8-kb mRNA, but it depends on the existence of pp24 [17, 18]. Recently, CAT gene was used as a reporter to verify that the enhancement of pp38 to the promoter depends on the existence of pp24 , it was further confirmed by the reporter gene of Enhanced Green Fluorescence Protein (EGFP) .
In order to compare the activity in both directions, and investigate whether the bi-directional promoter could be divided into two active promoters, a series of CAT plasmids were constructed by using the complete or divided promoters in two directions, and then transfected to the MDV infected CEFs. These different promoters activities were analyzed in transfected cells.
There is an uninterrupted 5-bp deletion in the promoter found in CVI988, which destroys a Sp1 site. The influence of the deletion to the bi-promoter was also studied in this work.
The complete bi-directional promoter activity in 1.8-kb mRNA direction is 15 times as that in pp38 direction
The CAT expression levels under the complete or divided promoters in opposite directions in uninfected, or rMd5-infected CEFs transfected with a set of CAT reporter plasmids
Complete or divided promoters in CAT reporter plasmids for transfection
3 ± 0
(3~3, n = 4)
4 ± 0
(4~4, n = 4)
4 ± 0
(4~4, n = 3)
4 ± 0
(4~4, n = 4)
4 ± 0
(4~4, n = 3)
4 ± 0
(4~4, n = 4)
4 ± 0
(4~4, n = 4)
3 ± 0
(3~3, n = 4)
52 ± 6.28
(41~60, n = 5)
34 ± 3.1
(29~39, n = 4)
781 ± 55.1 (704~842, n = 4)
19 ± 2.1
(16~23, n = 5)
54 ± 4.01
(47~68, n = 5)
635 ± 27.4 (587~700, n = 5)
The activity of the divided promoter decreased in both dierctions
The deletion of the Sp1 site in CVI988 causes the 20% activity decreased in 1.8-kb mRNA direction
It has been recognized for many years that there was a bi-directional promoter of about 300 bp between the transcriptional start sites of the pp38 gene and 1.8-kb mRNA transcripts [3, 4]. Beside two TATA boxes for gene transcription, the promoter contained several enhancer motifs including the Sp1, Oct1 and CAAT. In addition, a DNA replication origin and 17-bp reverse repeats were located within the promoter . It had reported that the bi-directional promoter activities in two opposite orientations were 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 . In our previous study, we reported that the heteropolymer pp38/pp24 could bind to the bi-directional promoter on their upstreams and regulate the promoter activity in expression of CAT or EGFP as reporter genes in transfected CEF [17–20].
To investigate whether the bi-directional promoter may be divided into two active single-orientation promoters, we cut up the promoter from site of -536 bp concerning on its symmetrical structure (Figure 4). The divided and intact promoters were cloned into the pCAT-Basic vector, respectively. In this vector, the inserted promoter activity could be quantitative analysized according to the CAT concentration in the transfected cells. The transfection indicated the activity of the divided promoters decreased in both orientations, especially in direction for 1.8-kb mRNA. It hints the bi-directional promoter is not only an assembly by two separate divided promoters, but also organized as a whole. Its entire activity is interrelated with the intact structure.
It was reported that CAT-activity expressed under the bi-directional promoter in the direction for 1.8-kb transcripts was significantly higher than that from the pp38 direction . The present study confirmed their result, and the promoter for the 1.8-kb mRNA transcripts is a much stronger promoter than that in the orientation for pp38.
Materials and reagents
pUC18 vector, T4 ligase, and all the enzymes were purchased from TaKaRa Biotechnology Co., Ltd (Dalian, China). Lipofectamine™ was purchased from Invitrogen (Beijing, China); plasmid purification Mini Kit was from Qiagen (Shanghai, China); pCAT-Basic vector was from Promega (Beijing, China); CAT ELISA detection Kit was from Roche (Shanghai, China); SPF chicken embryos were from SFAFAS Company (Jinan, China).
Cells and viruses
MDV rMd5 was rescued in culture from five cosmids containing a whole genome of parent virus Md5, which was kindly provided by Dr. Reddy S . This rescued rMd5 has a clear genetic background and predictable growth rate in CEF cells after its transfection. Eight distinct virulent MDV strains were used as templates to amplify the promoter regions: virulent strains (GA  and JM ), very virulent strains (Md5 , G2 (, and RB1B ), very virulent plus strain 648A  and vaccine strains (CVI988  and 814 ). All the strains were kindly provided from Dr. Cui Z. Z. These viruses were propagated in primary chicken embryo fibroblast (CEF) cells and inoculated with MDV-infected CEF at a 10:1 of CEF:virus-infected CEF ratio. The cell pellets were used for extraction of total genomic DNAs by proteinase K (Merck Co., Beijing, China) and phenol solutions as previously described.
Construction of recombinant plasmids expressing CAT gene under the control of different promoters
Primers used to generate a serial of plasmids to validate the activity of the promoter
The sites opposite to the ORF of pp38
Transfection of the CAT expressing recombinants to uninfected CEF and rMd5-CEF
Primary CEF cultures were prepared in a 60-cm2 flask until cells formed a monolayer and infected with rMd5-CEF stocks of 1×105 plaque form unit (PFU). The infected cell cultures were incubated for 3-4 days until cytopathogenic effect (CPE) was appeared in the monolayers. The MDV-CEF monolayers were trypsinized and the viable cell number was determined. 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). To prepare the secondary CEF monolayers, 1×106 cells were seeded into 35 mm dishes until cell monolayers formed 18-24 h later.
Transfection was carried out 18 h later when the secondary CEF monolayers were formed. Transfection of each recombinant plasmid DNA was performed by using LipofectAMINE™ reagent according to the manufacturer's instructions. Briefly, 2 μg plasmid DNA and 4 μl LipofectAMINE™ reagent were added into two separated polypropylene tubes with 100 μl of DMEM medium free of serum and antibiotic. These two solutions were mixed and incubated for 45 min at room temperature and then added into another 800 μl DMEM. A total of 1 ml of the transfection solution was carefully poured onto the cell monolayers in a 35 mm dish. After 8 h, 1 ml of complete medium with 10% bovine fetus serum were added to the transfected cell monolayers. All dishes were maintained at 37°C in a CO2 incubator. The expression of CAT was determined 48 h after transfection. The transfection on uninfected CEF was carried out as well as control.
Determination of CAT activity in transfected CEFs
Two days after transfection with plasmids pCAT-Basic (control), pPGA(pp38)-CAT, pPGA(1.8 kb)-CAT, pPCVI(pp38)-CAT, pPCVI(1.8 kb)-CAT, pdPGA(pp38)-CAT and pdPGA(1.8 kb)-CAT, the transfected CEF were harvested and resuspended in 500 μl lysis buffer (0.25 M Tris-HCl, pH7.0) per 35 mm dish. After 3 freeze-thaw cycles, samples were centrifuged for 5 min at 10,000 rpm. Aliquots (200 μl) of the supernatants were added into wells of 96-well ELISA plates to test CAT activity using CAT ELISA Kit (Roche, Cat.No.1363727). The concentration of the CAT in the lysates was measured using a calibration curve of known specific standards according to the manufacturer's instructions. Five replicates of transfections were carried out with 6 different CAT plasmid DNAs in each of rMd5-CEF or uninfected CEF cells. The significant differences among the groups were analyzed by student's test. The CAT activity in the pCAT-Basic transfected samples were also determined and analyzed as described.
This work is supported by the National Natural Science Foundation of China (grants 30700596). We are grateful for the advices and suggestions given by Dr. Cui Z Z (Animal Science and Technology College, Shandong Agricultural University, China) and for the critical review and suggestions of Dr. Xingquan Zhu.
- Addinger HK, Calnek BW: Pathogenesis of Marek's disease: early distribution of virus and viral antigens in infected chickens. J Natl Cancer Inst 1973, 50: 1287-1298.PubMedGoogle Scholar
- Bradley G, Hayashi M, Lancz G, Tanaka A, Nonoyama M: Structure of the Marek's disease virus BamHI-H gene family: genes of putative importance for tumor induction. J Virol 1989, 63: 2534-2542.PubMed CentralPubMedGoogle Scholar
- Bradley G, Lancz G, Tanaka A, Nonoyama M: Loss of Marek's disease virus tumorigenicity is associated with truncation of RNAs transcribed within BamHI-H. J Virol 1989, 63: 4129-4135.PubMed CentralPubMedGoogle Scholar
- Cui ZZ, Lee LF, Liu JL, Kung HJ: Structural analysis and transcriptional mapping of the Marek's disease virus gene encoding pp38, an antigen associated with transformed cells. J Virol 1991, 65: 6509-6515.PubMed CentralPubMedGoogle Scholar
- Jones D, Lee L, Liu JL, Kung HJ, Tillotson JK: Marek disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors. Proc Natl Acad Sci USA 1992, 89: 4042-4046. 10.1073/pnas.89.9.4042PubMed CentralView ArticlePubMedGoogle Scholar
- Xie Q, Anderson AS, Morgan RW: Marek's disease virus (MDV) ICP4, pp38, and meq genes are involved in the maintenance of transformation of MDCC-MSB1 MDV-transformed lymphoblastoid cells. J Virol 1996, 70: 1125-1131.PubMed CentralPubMedGoogle Scholar
- Ikuta K, Nakajima K, Naito M, Ann SH, Ueda S, Kato S, Hirai K: Identification of Marek's disease virus-specific antigens in Marek's disease lymphoblastoid cell lines using monoclonal antibody against virus-specific phosphorylated polypeptides. Int J Cancer 1985, 35: 257-264. 10.1002/ijc.2910350219View ArticlePubMedGoogle Scholar
- Nakajima K, Ikuta K, Naito M, Ueda S, Kato S, Hirai K: Analysis of Marek's disease virus serotype 1-specific phosphorylated polypeptides in virus-infected cells and Marek's disease lymphoblastoid cells. J Gen Virol 1987,68(Pt 5):1379-1389. 10.1099/0022-1317-68-5-1379View ArticlePubMedGoogle Scholar
- Maotani K, Kanamori A, Ikuta K, Ueda S, Kato S, Hirai K: Amplification of a tandem direct repeat within inverted repeats of Marek's disease virus DNA during serial in vitro passage. J Virol 1986, 58: 657-660.PubMed CentralPubMedGoogle Scholar
- Peng Q, Zeng M, Bhuiyan ZA, Ubukata E, Tanaka A, Nonoyama M, Shirazi Y: Isolation and characterization of Marek's disease virus (MDV) cDNAs mapping to the BamHI-I2, BamHI-Q2, and BamHI-L fragments of the MDV genome from lymphoblastoid cells transformed and persistently infected with MDV. Virology 1995, 213: 590-599. 10.1006/viro.1995.0031View ArticlePubMedGoogle Scholar
- Witter RL, Silva RF, Lee LF: New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: selected biological and molecular characteristics. Avian Dis 1987, 31: 829-840. 10.2307/1591039View ArticlePubMedGoogle Scholar
- Zhu GS, Ojima T, Hironaka T, Ihara T, Mizukoshi N, Kato A, Ueda S, Hirai K: Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification. Avian Dis 1992, 36: 637-645. 10.2307/1591759View ArticlePubMedGoogle Scholar
- Smith GD, Zelnik V, Ross LJ: Gene organization in herpesvirus of turkeys: identification of a novel open reading frame in the long unique region and a truncated homologue of pp38 in the internal repeat. Virology 1995, 207: 205-216. 10.1006/viro.1995.1067View ArticlePubMedGoogle Scholar
- Camp HS, Coussens PM, Silva RF: Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication. J Virol 1991, 65: 6320-6324.PubMed CentralPubMedGoogle Scholar
- Katsumata A, Iwata A, Ueda S: Cis-acting elements in the lytic origin of DNA replication of Marek's disease virus type 1. J Gen Virol 1998,79(Pt 12):3015-3018.View ArticlePubMedGoogle Scholar
- Shigekane H, Kawaguchi Y, Shirakata M, Sakaguchi M, Hirai K: The bi-directional transcriptional promoters for the latency-relating transcripts of the pp38/pp24 mRNAs and the 1.8 kb-mRNA in the long inverted repeats of Marek's disease virus serotype 1 DNA are regulated by common promoter-specific enhancers. Arch Virol 1999, 144: 1893-1907. 10.1007/s007050050713View ArticlePubMedGoogle Scholar
- Ding J, Cui Z, Lee LF, Cui X, Reddy SM: The role of pp38 in regulation of Marek's disease virus bi-directional promoter between pp38 and 1.8-kb mRNA. Virus Genes 2006, 32: 193-201. 10.1007/s11262-005-6876-2View ArticlePubMedGoogle Scholar
- Ding JB, Cui ZZ, Jiang SJ: The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus. Sci China C Life Sci 2006, 49: 53-62. 10.1007/s11427-004-0119-yView ArticleGoogle Scholar
- Ding J, Cui Z, Lee LF: Marek's disease virus unique genes pp38 and pp24 are essential for transactivating the bi-directional promoters for the 1.8 kb mRNA transcripts. Virus Genes 2007, 35: 643-650. 10.1007/s11262-007-0129-5View ArticlePubMedGoogle Scholar
- Ding J, Cui Z, Jiang S, Li Y: Study on the structure of heteropolymer pp38/pp24 and its enhancement on the bi-directional promoter upstream of pp38 gene in Marek's disease virus. Sci China C Life Sci 2008, 51: 821-826. 10.1007/s11427-008-0099-4View ArticlePubMedGoogle Scholar
- Reddy SM, Lupiani B, Gimeno IM, Silva RF, Lee LF, Witter RL: Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: use of a pp38 mutant to validate the technology for the study of gene function. Proc Natl Acad Sci USA 2002, 99: 7054-7059. 10.1073/pnas.092152699PubMed CentralView ArticlePubMedGoogle Scholar
- Eidson CS, Schmittle SC: Studies on acute Marek's disease. I. Characteristics of isolate GA in chickens. Avian Dis 1968, 12: 467-476. 10.2307/1588162View ArticlePubMedGoogle Scholar
- Schat KA, Calnek BW, Fabricant J, Abplanalp H: Influence of oncogenicity of Marek' disease virus on evaluation of genetic resistance. Poult Sci 1981, 60: 2559-2566.View ArticlePubMedGoogle Scholar
- Witter RL: Protection by attenuated and polyvalent vaccines against highly virulent strains of Marek's disease virus. Avian Pathol 1982, 11: 49-62. 10.1080/03079458208436081View ArticlePubMedGoogle Scholar
- Cui Z, Wei P, Ding J: Molecular comparisons of Marek's disease virus strains of different pathotypes for their gI, gE, pp38 and meq genes. J Shandong Agric Univ 2004.,35(1-5):Google Scholar
- Witter RL, Gimeno IM, Reed WM, Bacon LD: An acute form of transient paralysis induced by highly virulent strains of Marek's disease virus. Avian Dis 1999, 43: 704-720. 10.2307/1592740View ArticlePubMedGoogle Scholar
- Rispens BH, van Vloten H, Mastenbroek N, Maas HJ, Schat KA: Control of Marek's disease in the Netherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in laboratory vaccination trials. Avian Dis 1972, 16: 108-125. 10.2307/1588905View ArticlePubMedGoogle Scholar
- Tong GZ, Lin YH, Xun YW: Study on the immunity of Marek's disease: the cultivation and immunity assay for MDV vaccine strain. Chinese J Anim Poultry Infect Dis 1984, 15: 107-113.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.