The role of pp38 in regulation of Marek's disease virus bi-directional promoter between pp38 and 1.8-kb mRNA.
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.
Ding
Jiabo
J
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China.
Cui
Zhizhong
Z
Lee
Lucy F
LF
Cui
Xiaoping
X
Reddy
Sanjay M
SM
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis / Q000502:physiology*; D019677:Artificial Gene Fusion; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000821:virology; D019085:Fluorescent Antibody Technique, Direct; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D008381:Herpesvirus 2, Gallid / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; C071698:phosphoprotein pp38, Marek's disease virus; D015500:Chloramphenicol O-Acetyltransferase