The Role of pp38 in Regulation of Marek’s Disease Virus Bi-directional Promoter between pp38 and 1.8-kb mRNA

Abstract

Marek's disease virus (MDV) contains a bi-directional promoters located between pp38 gene and 1.8-kb mRNA in the long inverted repeat region of the viral genome. The involvement of pp38 gene in up-regulating the activity of these promoters was analyzed by transient expression of chloramphenicol acetyltransferase (CAT) reporter gene. Two CAT reporter plasmids, pP(pp38)-CAT and pP(1.8-kb)-CAT, were constructed to express CAT under the control of the bi-directional promoter in both orientations. These plasmids were transfected into chicken embryonic fibroblast (CEF), infected with rMd5 and pp38 deleted rMd5 (rMd5/Deltapp38), respectively. No CAT activity was detected in uninfected CEF as expected. CAT activities in rMd5/Deltapp38 virus infected CEF (rMd5/Deltapp38-CEF) were 3.5-fold lower using pP(pp38)-CAT and 12-fold lower using pP(1.8-kb)-CAT than those of the parental rMd5 infected CEF (rMd5-CEF). The significantly lower promoter activity in the pp38 deletion virus suggests that pp38 can regulate the activity of the bi-directional promoters, especially in the direction of 1.8-kb mRNA family. Co-transfection of pp38-expressing plasmid (pcDNA-pp38) into rMd5/Deltapp38-CEF significantly increased the activity of the bi-directional promoters using either pP(pp38)-CAT or pP(1.8-kb)-CAT. DNA mobility shift assay showed a binding of the 73-bp sequence of the bi-directional promoter with rMd5-CEF but not with rMd5/Deltapp38-CEF or uninfected CEF lysates. However, rMd5/Deltapp38-CEF lysates could bind the same 73-bp promoter sequence when co-transfected with pp38-expressing plasmid (pcDNA-pp38). All these data taken together suggest pp38 plays an important role in regulating the transcriptional activity of the bi-directional promoter.

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

“…A recombinant plasmid pP(1.8 kb)-CAT was constructed in a previous study according to published procedure [30]. To construct pBud-pp38-pp24 for co-expression of both pp38 and pp24, the complete pp38 gene was amplified from MDV GA strain and cloned into pBudCE4.1 vector (Invitrogen) at BamHI and SalI sites, and a recombinant plasmid pBud-pp38 was selected.…”

“…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].…”

“…The collected cells were lysed and the CAT activity was measured as reported [30]. Six to eight replicates of transfection were carried out for each group.…”

“…In addition, a DNA mobility shift assay showed a 73-bp fragment of the bi-directional promoter binds to parental rMd5-CEF or rMd5/Dpp38-CEF transfected with pcDNA-pp38, but not rMd5/Dpp38-CEF alone or CEF transfected with pcDNApp38. 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].…”