Ding Jiabo, Cui Zhizhong, Lee Lucy F, Cui Xiaoping, Reddy Sanjay M
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China.
Virus Genes. 2006 Apr;32(2):193-201. doi: 10.1007/s11262-005-6876-2.
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.
马立克氏病病毒（MDV）在病毒基因组的长反向重复区域中，于pp38基因和1.8 kb mRNA之间含有一个双向启动子。通过氯霉素乙酰转移酶（CAT）报告基因的瞬时表达，分析了pp38基因对这些启动子活性上调的影响。构建了两个CAT报告质粒，pP(pp38)-CAT和pP(1.8-kb)-CAT，以在双向启动子的控制下双向表达CAT。将这些质粒分别转染到鸡胚成纤维细胞（CEF）中，并分别用rMd5和缺失pp38的rMd5（rMd5/Deltapp38）进行感染。正如预期的那样，在未感染的CEF中未检测到CAT活性。与亲本rMd5感染的CEF（rMd5-CEF）相比，rMd5/Deltapp38病毒感染的CEF（rMd5/Deltapp38-CEF）中，使用pP(pp38)-CAT时CAT活性低3.5倍，使用pP(1.8-kb)-CAT时低12倍。pp38缺失病毒中显著较低的启动子活性表明，pp38可以调节双向启动子的活性，尤其是在1.8 kb mRNA家族的方向上。将表达pp38的质粒（pcDNA-pp38）共转染到rMd5/Deltapp38-CEF中，使用pP(pp38)-CAT或pP(1.8-kb)-CAT时，均显著增加了双向启动子的活性。DNA迁移率变动分析表明，双向启动子的73 bp序列与rMd5-CEF结合，但不与rMd5/Deltapp38-CEF或未感染的CEF裂解物结合。然而，当与表达pp38的质粒（pcDNA-pp38）共转染时，rMd5/Deltapp38-CEF裂解物可以结合相同的73 bp启动子序列。所有这些数据表明，pp38在调节双向启动子的转录活性中起重要作用。
