Paragraph: An oligonucteotide containing multiple AP-1 binding sites was introduced into the regulatory sequence in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV). Chloramphenicol acetyltransferase assay revealed that basal promoter activity of the mutated LTR was higher than that of the wildtype LTR in Crandell feline kidney (CRFK) cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the clone was transfected into CRFK cells. The virus production of the mutant in the cells was as high as that of the wildtype when determined by the reverse transcriptase activity assay. The growth of the mutant virus obtained from the transfected CRFK cells was examined in feline T lymphoblastoid cell lines (MYA-1 and FeL-039 cells) and primary feline peripheral blood mononuclear cells (fPBMCs). The growth was delayed when compared with that of the wild-type virus in all the cells used. Upon examination by polymerase chain reaction, the length of the LTR of the mutant virus was shortened in both MYA-1 cells and fPBMCs. Sequence analysis revealed that the insertion was completely deleted 39 days after infection in the MYA-1 cells. T. Miyazawa et al. several putative binding sites for enhancer proteins such as AP-1, AP-4, C/EBP are present [13, 14, 26] . Previously we reported that by deletion of AP-1 and AP-4 related sequences in the LTR, promoter activity was reduced in Crandell feline kidney (CRFK) andfelis catus whole foetus-4 (fcwf-4) cells using the chloramphenicol acetyltransferase (CAT) assay [11, 19] . Recently, Thompson et al. [29] reported that protein(s) present in the nuclear extract from feline T cells (F422 cells) which are permissive for FIV replication bind to the putative AP-1 and AP-4 binding sites. These findings suggest that the AP-1 in feline cells can bind to the AP-1 binding site in the L T R and is critical for the basal promoter activity of the LTR. Conversely, a m u t a n t virus which lacks the AP-1 and AP-4 binding sites replicated in feline T lymphoblastoid cell lines (MYA-1 and FeL-039 cells) [19] and primary feline peripheral blood mononuclear cells (fPBMCs) [17] . These data indicate that the AP-1 and AP-4 binding sites are not necessary for the efficient replication of FIV in feline T lymphocytes. In this study, we introduced an oligonucleotide containing multiple AP-1 binding sites adjacent to the original AP-1 binding site in the U3 region of the L T R and examined the effects of the insertion on the promoter and growth activities of the mutant. Cell culture CRFK cells [3] and fcwf-4 [9] were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated foetal calf serum (FCS). fPBMCs obtained from a specific pathogen-free cat were isolated as described previously [15, 16] . 21] and FeL-039 [30] cells, and fPBMCs were maintained in the RPMI 1640 growth medium supplemented with 10% FCS, antibiotics, 50 gM 2-merecaptoethanol, 2 g g/ml polybrene and 100 units/ml of recombinant human interleukin-2 at 37°C in a humidified atmosphere of 5% CO 2 in air. DNA constructs 
 Title: Effects of insertion of multiple AP-1 binding sites into the U3 region of the long terminal repeat of feline immunodeficiency virus
Output: True