key: cord-0004860-6do0r4en authors: Miyazawa, T.; Tomonaga, K.; Kawaguchi, Y.; Kohmoto, M.; Inoshima, Y.; Maedadel, K.; Mikami, T. title: Effects of insertion of multiple AP-1 binding sites into the U3 region of the long terminal repeat of feline immunodeficiency virus date: 1994 journal: Arch Virol DOI: 10.1007/bf01309453 sha: 00800dd5beb713ede24550797f2d2955389c2e51 doc_id: 4860 cord_uid: 6do0r4en An oligonucleotide 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 wild-type 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 wild-type 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. Feline immunodeficiency virus (FIV) is the etiological agent of an acquired immunodeficiency syndrome-like disease in cats [24, 32] . FIV is a member of the genus Lentivirus in the family Retroviridae [1] . Lentivirus expression is regulated by virally encoded proteins and by cellular transcriptional factors [2, 20] . Like other lentiviruses, FIV contains the rev gene [12, 25] , however it is not clear whether the virus contains a trans-activator gene [19, 28] . Also in the U3 region of FIV LTR, The CAT constructs under the control of the LTRs of FIV, termed p SPTM 1CATand pSPTM 1A PvCAT, were described previously [10, 19] . They were made by placing FIV LTR from an infectious clone ofFIV TM1 strain (pFTM191CG) [14] and its deleted mutant in front of the CAT gene ofpSPCAT which has both CAT gene and poly A signal [19] . An infectious clone of FIV, termed pSTM2, and a mutated infectious clone which lacks a 31 base pair fragment containing one AP-1 and one AP-4 binding sites, termed pSTM2D2, were described previously [19] . The pSTM2 5' and 3' LTRs were derived from TM2 and TM1 strains of FIV, respectively [19] . After digestion of pSTM2 with Sad, a small fragment which contained the 5' LTR ofpSTM2 was subcloned into the SacI site of p SP72 vector (Promega, Madison, WI, U.S.A.) and designated as pSTM2LTR. To introduce an oligonucleotide containing multiple AP-1 binding sites into pSTM2LTR, oligonucleotides (5'-CTAGCATGAGTCAGAGTFATGAGTCAGAGT-3' and 5'-CTAGACTCTGA-CTCATAACTCTGACTCATG-3') were synthesized. The oligonucleotides were annealed, phosphorylated by T4 DNA kinase and then ligated into NheI site ofpSTM2LTR, and designated as pSTM2-5APLTR. The sequence of the pSTM2-5APLTR was determined by the cycle sequencing method using dye-terminator supplied by Applied Biosystems (ABI) (Foster City, CA, U.S.A.). To generate pSTM2 (5AP, 5') which has the mutated LTR at the 5' end of the FIV genome ofpSTM2, the SacI fragment ofpSTM2 which contains the 5' LTR was substituted with the SacI fragment ofpSTM2LTR-5AP. To generate pSTM2-5AP which has the mutated LTR on both sides of the genome, a BamHI fragment of the pSTM2 (5AP, 5') was introduced into the BamHI site ofpSTM2-5APLTR. To construct pSTM2-5APCAT, the SacI-NarI fragment ofpSTM2-5APLTR was cloned into the pSPCAT, pVisLTRCAT [7] which is a visna virus LTR CAT reporter plasmid was kindly provided by Dr. J. E. Clements (The Johns Hopkins Univeristy, MD, U.S.A.). For transfection of plasmid DNA into CRFK and fcwf-4 cells, cells were plated in six-well dishes one day before transfection. Plasmid DNAs were transfected by the calcium phosphate coprecipitation method [6, 31] . Four hours after transfection, the cells were washed with phosphatebuffered saline (PBS), shocked with glycerol and then placed in fresh medium. For the CAT assay, cell monolayers in each well of six-weU dishes were harvested by scraping 48 h after transfection. After being washed once with PBS, the cells were tysed by freezing and thawing four times in 250 mM-Tris-HC1 pH 7.8. Cell debris was pelleted by centrifugation for 5 min at 4 °C and various amounts of each extract were assayed for CAT activity [5] by the solvent partition method [22] . In brief, a 240 gl reaction mixtu're containing 100 mM-Tris-HC1 pH 7.8, 1.0 mMchloramophenicol, 3.7 kBq of [14C] acetylcoenzyme A (Du Pont, NEN), and cell extract was overlaid with 5 ml of scintillation fluid (EconofluorII; Du Point, NEN). The reaction was carried out at 37 °C and production of radioactively labeled acetylchloramphenicol was monitored by counting in a liquid scintillation counter. The CAT activity of each promoter plasmid was presented as the net d.p.m, of product formed/h. All the CAT assay data reported in this paper are from the points in the linear range of the assay. MYA-1 and FeL-039 cells, and fPBMCs (1.5 x 106 cells) were infected with the FIVs derived from the infectious molecular clones. The cells were seeded at 3 x 105 cells/ml in growth medium. The cell numbers were adjusted to 3 x 105l ml in tYesh growth medium at the indicated time. The Mg2+-dependent RT activity in cell culture supernatants was assayed as described previously [23] . For amplification ofFIV LTRs, an antisense primer (N 1 ) (5'-GTCCCTGTFCGGGCGCCAACT-Y, nucleotide 381-361) and a sense primer (N2) (5'-GATGGCAAATCTAGAGAACCGC-Y, nucleotide 9011-9032) were synthesized corresponding to the primer binding site and an upstream sequence from the polypurine tract of FIV, respectively. The sequences of primers originated fi'om the sequence of FIV TM2 strain [13] . PCR was carried out by the method of Saiki et al. [27] in a 50 gl volume overlaid with an equal volume of mineral oil. A GeneAmp PCR Reagent kit (Perkin Elmer Cetus, Norwalk, CT U.S.A.) was used for the reactions. Amplification proceeded for 30 cycles in DNA thermal cycler model P J2000 (Perkin Elmer Cetus, Norwalk, CT, U.S.A). One cycle consisted of incubations at 94, 64, and 72 °C for 1, 1, and 2 rain, respectively. After amplification, a 5 gl sample of the 50 gl-reaction was electrophoresed on a 1.5% agarose gel (in Tris-borate-EDTA but~er). Amplified DNA digested with XbaI and NarI was subcloned into pUC118 and then used for sequence analysis. The double stranded DNA was annealed with N1 or N2 primer, extended by the cycle sequencing method using dye-terminator supplied by ABI, and then analyzed by a model 370A ABI autosequencer. Oligonucleotides containing two AP-1 sites were synthesized and inserted into the NheI site of FIV LTR (pSTM2LTR) to generate pSTM2-5APLTR. Sequence analysis revealed that the pSTM2-5APLTR has five AP-1 binding sites (Fig. 1A) . The insertion of the plasmid was stable in E. coli. The mutated LTR containing five AP-1 sites was placed upstream from the CAT gene and poly A signal ofpSPCAT to construct pSTM2-5APCAT. The sequence of the U3 region of pSPTM1CAT, pSPTM1A PvCAT and pSTM2-5APCAT is shown in Fig, 1A , and the schematic view of the LTR CAT reporter plasmids is shown in Fig. 1B . The LTR of TM2 strain differed from that of TM 1 strain only at positions -138 (G to A) and -159 (T to C) [13, 14] , and both the LTRs of TM1 and TM2 showed almost the same promoter activity in fcwf-4 and CRFK cells (unpubl. data). Two ~tg of pSTM2-5APCAT, pSPTM1CAT, pSPTM1APvCAT, pSPCAT or pVisLTRCAT were transfected into CRFK and fcwf-4 cells. Forty-eight hours after transfection, the CAT products of the clones were measured by the solvent partition method. The basal promoter activity of the pSTM2-5APCAT was significantly higher than that of the pSPTM 1 CAT in CRFK cells ( Fig. 2A) 2B ). In addition, the activity of the pSTM2-5APCAT was lower than that of the pVisLTRCAT in both cell types (Fig. 2) . The mutated LTR was introduced into an infectious molecular clone of FIV (termed pSTM2) and designated pSTM2-5AP. Five gg of pSTM2 (wild-type), pST-M2D2 (AP-l-deleted mutant) or pSTM2-5AP were transfected into CRFK cells, and three days after transfection, the virus production in the culture supernatant was measured using the RT activity assay. As shown in Fig. 3 , the RT activity of the pSTM2-5AP was similar to those of pSTM2 and pSTM2D2. Samples of the culture supernatants containing equivalent radioactivity from the CRFK cells transfected with the respective infectious molecular clones were inoculated into MYA-1 cells, FeL-039 cells and fPBMCs, and the virus production was monitored by the RT activity assay. As shown in Fig. 4 , the growth of the virus derived from pSTM2-5AP was delayed in all the cells, however, the peaks of the RT activities of both viruses occurred at similar levels in either MYA-1 (Fig. 4A) or FeL-039 (Fig. 4B) cells. On the other hand, the peak of RT activity of the virus from pSTM2-5AP was lower than that of the pSTM2 in fPBMCs (Fig. 4C) . The culture supernatant of MYA-1 cells infected with the virus from pSTM2-5AP at 19 days after infection (Fig. 4A ) was transferred onto MYA-1 cells and fPBMCs, and the virus production was monitored by the RT activity assay (Fig. 5) . In MYA-1 cells (Fig. 5A) , the growth of the virus from pSTM2-5AP was similar to that of the virus from pSTM2. On the other hand, in fPBMCs (Fig. 5B) , the growth rate of the mutant was lower than that of the virus from pSTM2. To determine the stability of the insertion of the multiple AP-1 binding sites during the infection experiments ( Fig. 4A and C, Fig. 5 fPBMCs harvested at final sampling times and were amplified by PCR. As shown in Fig. 6 , the length of the mutant LTR was shortened in both MYA-1 cells and fPBMCs harvested at different times. Partial nucleotide sequences of the PCR products obtained from the MYA-1 cells harvested at 29 days after infection (Fig. 4A ) and 20 days after infection (Fig. 5 ) were compared with those of the U3 region of plasmids pSPTM1 (TM1) and pSTM2-5APLTR (TM2-5AP). The same sequence with the pSTM2-5APLTR and three types of deleted sequences (del. 1-3 in Fig. 7) were obtained from the MYA-1 cells infected with the FIV derived from CRFK cells transfected with pSTM2-5AP, and harvested at 29 days after infection (Fig. 4A) . The sequence ofdel. 1 has two AP-1 binding sites and those ofdel. The LTRs of the lentiviruses contain many binding sites for cellular transcriptional factors. FIV, visna virus and caprine arthritis-encephalitis virus (CAEV) possess putative binding sites of AP-1, AP-4 and C/EBP in the LTRs [7, 8, 20] . Of these, AP-1 a n d / o r AP-4 binding sites have been shown to be critical for efficient transcription ofvisna virus [8] and FIV [11, 19, 28] . Although FIV has only one AP-1 binding site in the U3 region of the LTR [14, 26] , visna virus and CAEV have multiple AP-1 binding sites in this region [7] . In addition, we found that the promoter activities ofvisna virus and CAEV LTR are rather higher than that of the FIV LTR in CRFK and SW480 (human colon carcinoma) cells [18] . It has been suggested that the high promoter activities of the visna virus and CAEV are partly due to the multuiple copies of the AP-1 binding sites in the LTRs. In this study, by insertion of multiple AP-1 binding sites adjacent to the original AP-1 binding site in the enhancer region of FIV LTR, significant enhancement of promoter activity was observed in CRFK cells. These data suggested that the insertion is effective at least in these cells. However, the activity of the mutant LTR was still lower than that of visna virus LTR. It might be possible that the high promoter activity of visna virus cannot be attributed only to the multiple copies of the AP-1 binding sites. In contrast to the CRFK cells, in fcwf-4 cells no significant enhancement of promoter activity was observed by the insertion, while by deletion of an AP-1 site together with one AP-4 site out of two, the promoter activity was significantly reduced in the cells. The reason for the discrepancy is unknown at present, however it is possible that the promoter activity in fcwf-4 cells is less dependent on the AP-1 site than the deleted AP-4 site. To examine whether the insertion has positive effect on the replication capability of FIV, we constructed an infectious molecular clone which contained mutated LTRs on both sides of the genome. Firstly, the mutant infectious clone was transfected into CRFK cells, and the transient virus production of the mutant was 46 T. Miyazawa et al. compared with that of both the wild-type and the AP-1 deleted mutant. It was expected that the virus production would correlate with the results of the CAT assay. However, no significant difference was observed among these infectious clones. We previously demonstrated the presence of suppressor gene-like activity of FIV which was similar to the nefgene activity of primate lentiviruses and reduced the promoter activity of the LTR [19] . It is possible that the suppressor gene-like activity abrogated the enhancement and reduction of promoter activity caused by the insertion and deletion, respectively. Next, the virus production of the mutant infectious clone was compared with that of the wild-type in feline T lymphocytes. The growth of the mutant virus was delayed in MYA-1 cells, FeL-039 cells and fPBMCs. In addition, when the mutated virus harvested from MYA-1 cells at 19 days after infection (Fig. 4) was transferred onto normal MYA-1 and fPBMCs, the growth of the mutant virus had changed to the level of the wild-type FIV in the MYA-1 cells (Fig. 5A) . PCR analysis to amplify the LTR in the cultures revealed that the length of the LTR was shortened to be similar to that of the wild-type virus. Sequence analysis revealed that the insertion of the multiple AP-1 binding sites was completely deleted. These data indicate that the multiple AP-1 binding sites introduced into the LTR have a negative effect on the viral growth activity in feline T lymphocytes, and deletions had occurred by 19 days. Previously we also reported that the AP-1-deleted mutant grew as well as the wildtype in MYA-1 and FeL-039 cells [19] and primary fPBMCs [17] , and concluded that the AP-1 site is not required for the replication of FIV in feline T lymphocytes. The mechanism of the delay of the viral growth of the AP-1 inserted mutant in feline T lymphocytes is unclear at present. However, since the AP-1 binding site (TGA[G/C]TCA) contains palindrome sequence [4] , it is possible that multiple AP-1 binding sites introduced in the LTR readily form secondary structures at mRNA level and inhibit reverse transcription from full length genomic RNA to complementary DNA in feline T lymphocytes. Visna virus and CAEV have multiple AP-1 binding sites, however most of the AP-1 sites in both viruses are slightly different from the consensus sequence and are present dispersedly [7, 8] . Therefore it is unlikely that the mRNAs of the U3 region of these viruses form strong secondary structures. Nevertheless, the present study together with the previous studies [17, 18] demonstrates that there is no linear relationship between the promoter activity' of the virus determined by the CAT assay and the viral replication capability, therefore to understand the significance of the enhancer and promoter binding sites regarding the virus life cycle, it is necessary to construct infectious clones which lack other binding sites and to compare the growth rates of the mutant viruses. Classification and Nomenclature of Viruses. Fifth Report of the International Committee on Taxonomy of Viruses Regulatory pathways governing HIV-1 replication Development, characterization, and viral susceptibility of feline (Felis ca tus) renal cell line (CRFK) Fos and Jun: the Ap-1 connection Recombinant genomes which express chloramphenicol acetyltransferase in mamalian cells A new technique for the assay of infectivity of human adenovirus 5 DNA Nucleotide sequence and transcriptional activity of the caprine arthritisencephalitis virus long terminal repeat Sequences in the viana virus long terminal repeat that control transcriptional activity and respond to viral trans-activation: involvement ofAP-1 sites in basal activity and trans-activation Expression of feline infectious peritonitis coronavirus antigens on the surface of feline macrophage-like cells Activation of feline inmmnodeficiency virus long terminal repeat by feline herpesvirus type 1 Sequences within the feline immunodeficiency virus long terminal repeat that control transcriptional activity and respond to activation by feline herpesvirus type 1 Identification of the feline immunodeficiency virus rev gene activity Molecular characterization and heterogeneity of feline immunodeficiency virus isolates Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U.S. isolate Preliminary comparisons of the biological properties of two strains of feline immunodeficiency virus (FIV) isolated in Japan with FIV Petalurna strain isolated in the United States Establishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus Growth properties of a feline immunodeficiency virus mutant which lacks an AP-1 binding site in primary peripheral blood mononuclear cells FIV with five AP-1 binding sites Comparative functional analysis of the various lentivirus long terminal repeats in human colon carcinoma cell line (SW480 cells) and feline renal cell line (CRFK cells) The AP-I binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes The genome of feline immunodeficiency virus Further characterization of a feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive for feline immunodeficiency virus A novel rapid assay for chloramphenicol acetyltransferase gene expression Isolation of simian immunodeficiency virus from African green monkeys and seroepidemiologic survey of the virus in various non-human primates Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome Elder JH (1192) Identification of the Rev transactivation and Rev-responsive element of feline immunodeficiency virus Comparison of two host cell range variants of feline immunodeficiency virus Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Regulation of gene exepression directed by the long terminal repeat of the feline immunodeficiency virus Cis-and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat Altered cell tropism and cytopathicity of feline immunodeficiency viruses in two different feline CD4-positive, CD8-negative cell lines DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats We thank Dr. S. Tokiyosi (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan), Dr. K. Ikuta (Institute of Immunological Science, Hokkaido University, Sapporo, Japan), and Dr. M. Hattori (Kyoto University, Kyoto, Japan) for the supply of the oligonucleotides, FeL-039 ceils and recombinant human interleukin-2 producing Ltk~L-2.23 cells, respectively. We also thank Dr. J. E. Clements (The Johns Hopkins University, Maryland, U.S.A.) for providing FIV with five AP-1 binding sites 47 pVisLTRCAT. We are especially grateful to Dr. J. MacDonald (University of Glasgow, Glasgow, U.K.) for helping in the preparation of the manuscript. This study was supported in part by grants from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare of Japan. Y. Kawaguchi is supported by the Recruit Scholarship. Authors' address: Dr. T. Mikami, Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan.Received April 13, 1994