Comparison Studies On Transcriptional Regulation Of Donkey Leukocyte Attenuated Equine Infectious Anemia Vaccine Virus And Its Parental Virus

Two recombinant plasmids containing genes encoding the trans-activate protein (Tat) of EIAV were gained by using RT-PCR ampliing RNAs extracted from donkey leukocyte attenuated EIAV (DLA EIAV).These clones were designated as Etai3 19 and Etat3 12, respectively. The entire tat gene was then amplified from a conservative clone as template after sequence comparison. The tat gene was 231 bp in length, encoding a protein of 76 amino acids. Subcloning of the tat into plasmid pcDNA3 generated a recombinant expression plasmid tat-pcDNA3, which has been proved to be trans-active through in vitro transient transfection. The promoting activities of three long terminal repeats (LTRs) from donkey-adapted equine infectious anemia virus (D-A EIAV), DLA EIAV and EIAV strain Wyoming were comparatively studied in donkey leukocytes by driving chloroamphenicol acetyltransferase (CAT) expression. The results indicated that the promoting activity of LTR from DLA EIAV was slightly higher than that of 0-A EIAV, and lower than strain Wyoming. The CAT activity was greatly increased when the cells were cotransfected with recombinant plasmid tat-pcDNA3. The CAT activity was increased 4.8 folds driving by LTR of D-A EIAV, 6.0 folds with DLA EIAV and 3.2 folds with strain Wyoming when cotransfected with recombinant plasmid tat-pcDNA3. In order to determine the function of GATA motif, which is unique in Chinese EIAV (0-A and DLA EIAV )enhancer unit among U3 region, the GATA transcription factor binding motif TGATAA has been changed to TTCGAA by PCR-directed mutagenesis to abolish its binding. Then the promoting activities of U3+R with or without the GATA motif were examined by CAT assays in K562 cell line. The results showed that the activities of U3+R region were decreased by 43% with 0-A EIAV and 52% with DLA EIAV after this mutation. Above results suggested that the GATA motif might play a positive role during the transcriptional regulation of Chinese EIAV (0-A EIAV and DLA EIAV). This result is also consistent with the observations from other retrovirus expression regulation. In addition, two different bases in 0-A EIAV TAR element were mutated to the same as those of DLA EIAV TAR by PCR-directed mutagenesis. Namely, two adenines (A) located within 0-A EIAV TAR element and its neighbor region, respectively, were mutated to guanine (G) and thymine (T). CAT activity analysis showed that this mutation failed to bring any significant change to promoting activity of 0-A U3+R region, which suggested that the base mutation in DLA TAR element might not change its secondary structure in donkey leukocyte, or a bugle might form in DLA EL4V TAR element but could not be recognized by ELAV Tat protein because of its appearance in the terminal of stem structure.