The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus

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(1.8-kb)-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 rMd5deltapp38 infected CEF (rMd5deltapp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF, and no EGFP could be detected in uninfected CEF or rMd5deltapp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5deltapp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5deltapp38-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.

Marek’s disease virus unique genes pp38 and pp24 are essential for transactivating the bi-directional promoters for the 1.8 kb mRNA transcripts

“…No expression was detected in cells co-transfected with either pp38 or pp24 alone [31]. These results taken together indicate that coexpression of pp38 and pp24 transactivates the bi-directional promoter resulting in expression of CAT or the EGFP, but transactivation does not occur with expression of pp38 or pp24 alone.…”

“…Based on the previous study, the 73-bp fragment of subregion ||(from base -514 to -442) in the promoter was the only fragment retarded by pp38-associated proteins from MDV-CEF or pp38 and pp24 together from transfected CEF [30,31]. To further identify the precise sequence bound by pp38-pp24, double-stranded DNA samples of small overlapping subfragments covering the 73-bp fragment (#1: -514 to -490; #2: -500 to -457; #3: -467 to -442) were synthesized by a commercial service (Takara Biotechnology (Dalian) Co., Ltd).…”

“…In plasmid pBud-pp38-pp24, the pp38 and pp24 were expressed under the control of promoters P CMV and P EF-1a in the expressing vector pBudCE4.1, respectively. Two additional plasmids, pcDNA-pp38 and pcDNA-pp24, were constructed by using the pcDNA-3.1/Zeo(+) vector (Invitrogen) as described previously [30,31]. All of the above plasmids were purified with a QIA prep Spin Miniprep Kit (Qiagen).…”

“…For each transfection, we used 2 lg of plasmid DNA and 4 ll of LipofectAMINETM reagent (Gibco BRL), and the transfection was stopped after 8 h. Detailed procedures are given in previous reports [30,31].…”

“…This indicates that pp38 may be necessary for regulating the bi-directional promoter [30]. In a separate study, we co-transfected CEF with both pcDNA-pp38 and pcDNA-pp24 expressing plasmid DNA and showed that the expression of green fluorescence protein gene (EGFP) was under the control of the bi-directional promoter [31]. The present study demonstrates the transactivation activity of both pp38 and pp24 together on the bi-directional promoter and identifies a seven base binding motif using the gel mobility shift assay.…”