JOURNAL OF VIROLOGY, JUlY 1989, p. 3001-3015 Vol. 63, No. 7 0022-538X/89/073001-15$02.00/0 Copyright ©) 1989, American Society for Microbiology

Sequences in the Visna Virus Long Terminal Repeat That Control Transcriptional Activity and Respond to Viral trans-Activation: Involvement of AP-1 Sites in Basal Activity and trans-Activation

JAY L. HESS,' JUDY A. SMALL,2 AND JANICE E. CLEMENTS'2.3* Departments of Comparative Medicine,3 Molecuilar Biology and Genetics,' and Neutrology,2 The Jo/ins Hopkins University School of Medic ine, Baltimore, Maryland 21205 Received 21 October 1988/Accepted 31 March 1989

Visna virus is a pathogenic lentivirus of sheep whose gene expression is developmentally regulated in cells of the monocyte-macrophage lineage. Gene expression directed by the visna virus long terminal repeat (LTR) is increased in infected cells by a virus-encoded trans-acting protein. trans-Activation is mediated in part by increases in the steady-state level of mRNA. Deletion and linker-scanner mutants were constructed to locate sequences in the LTR that regulate transcription and are responsive to viral trans-activation. The activities of these mutants were tested by using them to drive transcription of the bacterial gene for chloramphenicol acetyltransferase in transient expression assays. Three regions located between -140 and the cap site were found to be important for basal transcriptional activity, and the importance of each region was found to be dependent on the cell type. Sequences responsive to viral trans-activation were found to be the same sequences required for basal transcriptional activity. The visna virus LTR contains six sequences that are homologous to the recognition site for cellular transcriptional factor AP-1 and a single sequence homologous to the recognition site for transcriptional factor AP-4. Both of these classes of binding sites appear to be important for regulating the basal level of transcription of visna virus. The AP-1-binding site most proximal to the TATA box was found to be one target for viral trans-activation. The visna virus promoter was found to be activated by serum; this serum response has also been mapped to the AP-1-related sequences in the LTR.

Lentiviruses are a subfamily of nononcogenic retroviruses that increases transcription from the viral LTR (22, 27, 29, that cause chronic diseases with unusually long incubation 46, 50) and may also act posttranscriptionally to increase periods. Visna virus, the prototype of this group, causes viral gene expression (9, 14, 41, 51, 67). The second gene pneumonitis and a progressive demyelinating disease in product, rev,, is a 19- to 20-kilodalton protein that increases sheep months to years after the initial infection (56, 57). The expression of the structural genes gag and env (14, 30, 59). target cells for visna virus in infected animals are monocytes In the studies reported here, deletion and linker-scanner and monocyte precursors in bone marrow and spleen (17). mutations in the LTR of visna virus were used to identify Visna virus replication is restricted in vivo so that infected sequences important for transcriptional activity. The activi- monocytes do not express significant levels of viral RNA or ties of these mutant LTRs were measured by their abilities to protein until they mature into macrophages in the lungs, drive transcription of the cat gene in a variety of cell types. joints, and brain (16, 17, 44). In contrast to its restricted In addition, the activities of some of these mutants were replication in vivo, visna virus replicates to high titers in measured in cells grown at both high and low serum concen- fibroblasts in culture, ultimately killing the cells. This high trations to locate sequences mediating the responsiveness of replication rate and marked cytopathic effect are important the promoter to serum. These experiments indicate that features that distinguish lentiviruses from other retroviruses. three regions in the visna virus LTR are important for basal The high replication rate of visna virus and other lentivi- transcriptional regulation. The first of these sequences is the ruses, such as human immunodeficiency virus (HIV), can be TATA box. The second is the recognition site for the attributed in part to the production of virally encoded recently described transcriptional factor AP-4 (40), which is that increase the of viral trans-acting proteins expression present as a single copy in the viral LTR. The third sequence genes. The genomes of both visna virus and HIV contain is the recognition site for cellular transcriptional factor AP-1 the and enm additional open reading frames between pol (6, 7, 33, 34), which is present in multiple degenerate copies genes that are unique to lentiviruses (18, 19, 49, 62, 66). in the visna virus LTR. Of these multiple AP-1 sites, the These open reading frames, which have been found in all of copy proximal to the TATA box, which has a sequence the lentiviruses sequenced, are expressed as small (1.5- to identical to the AP-1 consensus, appears to be critical for 2-kilobase) spliced transcripts that are involved in the regu- efficient transcription. lation of viral gene expression (3, 10, 11, 13, 24, 54, 55, 60, To investigate the mechanisms of visna virus trans-acti- 61). Recently, we have identified a gene in the visna virus determine whether the factors func- genome that is analogous to the tat gene of HIV and is vation and trans-acting responsible for trans-activation of the visna virus long ter- tion at the transcriptional level, primer extension experi- ments were done. The levels of RNA were minal repeat (LTR) (10). In HIV, two gene products have steady-state in uninfected and visna virus-infected cells trans- been identified that are encoded by overlapping reading quantitated with the visna virus LTR driving frames. The tat gene encodes a 14- to 15-kilodalton protein fected plasmids containing transcription of the cat gene. These experiments showed that at least 50% of visna virus trians-activation is mediated * Corresponding author. by increases in the steady-state level of mRNA. 3001 3002 HESS ET AL. J. VIROL.

To identify sequences recognized by the visna virus phoresis. The purified plasmids were religated with T4 DNA trans-acting factor, deletion and linker-scanner mutants of ligase and transformed into DH-5 cells. Large-scale plasmid the visna virus LTR were used in a series of experiments on preparations were made from 500-ml cultures directly inoc- uninfected and infected goat synovial membrane (GSM) and ulated with the transformation cultures for each time point. sheep choroid plexus (SCP) cells. The data suggest that the Following purification on CsCl gradients, the plasmids were sequences required for trans-activation are the same as digested with XhoI and KpnI and run on 2% agarose gels, those required for basal activity in uninfected cells. tracns- yielding a range of fragments between 250 and 550 base pairs Activation could not be totally eliminated without abolishing (bp) long (corresponding to deletions of up to 300 bp in from promoter activity; however, some mutants showed reduced the EcoRV site). Fragments of various lengths were electro- responsiveness to trans-activation. These mutations either eluted from the agarose gels and ligated into a modified deleted or overlapped sequences that are homologous to the version of pVIS-LTR-CAT (the KpnI site upstream from the recognition site for cellular transcription factor AP-1. The viral insert in pVIS-LTR-CAT was destroyed, and then the abilities of these AP-1-related sequences to respond to HindlIl site at the 3' end of the viral insert was converted to trans-activation in visna virus-infected cells were tested by a KpnIl site by using synthetic linkers) that was cut with XhoI using plasmids containing synthetic copies of the AP-1 and KpnI. One 3' deletion mutant ending at +22, which was recognition sequence adjacent to the herpes simplex virus tk used in making the +22/+30 linker-scanner mutant, was promoter, which by itself responds poorly to trans-activa- prepared by exonuclease III digestion of a different LTR tion (2). The activities of AP-1-tk hybrid constructs were construct that contained additional 3' sequences extending increased in infected cells, indicating that the AP-1 sequence into the gag gene. The only difference resulting from using is probably one target for visna virus trans-activation. this mutant was that it introduced the sequence GGGT ACCC rather than _CGGTACCC into the +22/+30 linker- MATERIALS AND METHODS scanner mutant (see below). Plasmids prepared from indi- vidual colonies were analyzed by restriction digests and by Construction of 5' deletion mutants. Construction of plas- double-stranded DNA sequencing with primers complemen- mid pVIS-LTR-CAT, which contains the entire visna virus tary to either the 5' end of the cat gene or various locations LTR driving transcription of the cat gene, has been previ- in the viral U3 region (for the complete set of 3' deletion ously described (25). To generate deletions at the 5' end of mutants, see Fig. 3). the visna virus LTR, pVIS-LTR-CAT was digested with Construction of internal-deletion and linker-scanner muta- BstEII, an enzyme which cuts at a unique site within the U3 tions. Internal-deletion and linker-scanner mutations (39) region (see Fig. 1). Linearized DNA (100 pLg) was incubated were constructed by digesting sets of 5' and 3' deletion with 5.6 U of nuclease BAL 31 (New England BioLabs, Inc.) mutants of pVIS-LTR-CAT with KpnI (which cuts at the at 40°C, and 10-pg samples were removed at 1-min intervals. deletion endpoint) and BglI (which cuts in the ampicillin The nuclease reactions were stopped with EGTA [ethylene gene). The appropriate restriction fragments were purified glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid], on agarose gels, ligated together, and transformed into the plasmids were precipitated with ethanol, and the ends of bacteria. The internal deletion mutant +22/+30 was con- the nuclease-treated plasmid were made flush by treatment structed by cleaving pVIS-LTR-CAT with EcoRV and SstI, with the Escherichia coli DNA polymerase Klenow frag- treating it with DNA polymerase I to generate flush ends, ment. The plasmids were reprecipitated with ethanol and and religating the plasmid following separation from the then digested with SmaI, which cuts at a unique site in the small fragment on agarose gels. The +51+1 mutant is actu- vector 5' to the visna insert, generating flush ends. The ally a linker insertion mutant in which 12 additional bases SmaI-digested plasmids were purified away from the small were inserted 5 bp downstream from the cap site. Plasmids fragments generated by using agarose gel electrophoresis. prepared from individual colonies were analyzed by restric- The purified plasmids were ligated with T4 DNA ligase and tion digests, and all constructs were confirmed by sequenc- transformed into E. coli DH-5. DNA prepared from individ- ing across the mutated region in each construct (for the ual colonies was analyzed by restriction digests, and the boundaries of each region confirmed by sequencing, see the extent of each deletion was determined by sequencing each vertical lines in Fig. 6). plasmid by using a primer complementary to sequences Transfections and CAT assays. Nearly confluent monolay- adjacent to the SmaI site of pVIS-LTR-CAT (5, 53; for the ers of sheep choroid plexus (SCP) cells (43), sheep alveolar complete set of 5' deletion mutants, see Fig. 2). macrophages (15), goat synovial membrane (GSM) cells (42), Construction of 3' deletion mutants. Deletion mutant pVIS- or mouse L cells (from ATCC) grown in 35-mm-diameter LTR-CAT(+91) was constructed by digesting pVIS-LTR- dishes were transfected by the DEAE-dextran technique (37) CAT with EcoRV, which cuts at a unique site in the R region coupled with a dimethyl sulfoxide shock as previously (see Fig. 1), and ligating on Hindlll linkers, followed by described (25, 36). SCP or GSM cells to be infected were HindlIl digestion and purification of the plasmid on an infected with visna virus strain 1514 at 40 to 50 hours before agarose gel. The purified plasmids were then religated and transfection at a multiplicity of approximately 1 50% tissue transformed into bacteria. To generate other deletions at the culture infective dose per cell. Depending on the experi- 3' end of the visna virus LTR, pVIS-LTR-CAT was digested ment, cells were maintained on modified Eagle medium with EcoRV, the linearized DNA (360 pg) was incubated containing 0.5, 2, or 5% fetal bovine serum (FBS) following with 160 U of exonuclease III (Pharmacia) at 23°C, and 30-[Lg transfection. After one rinsing with diluent, cell monolayers samples were removed at 2-min intervals. The samples were were harvested for a chloramphenicol acetyltransferase incubated at 68°C to inactivate the nuclease and then treated (CAT) assay by scraping at 48 h after transfection. After with mung bean nuclease (Pharmacia). Following phenol- being washed once with cold phosphate-buffered saline (pH chloroform and chloroform extractions, the plasmids were 7.4), the cells were lysed by freeze-thawing three times in 60 ethanol precipitated and ligated to KpnI linkers (Pharmacia). pd of 250 mM Tris chloride (pH 7.8). Cell debris was pelleted The plasmids were reprecipitated, digested with KpnI, and by centrifugation, and various amounts of the extracts were separated from the small fragments by agarose gel electro- assayed as previously described (21, 25). The percent acet- VOL. 63, 1989 MUTATIONAL ANALYSIS OF THE VISNA VIRUS LTR 3003

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------. 00-- AP-1AP-1AP-1 AP4 -- -AP-1- TA +1Fr U3 | 43 4 TATA / / U5 Bst E 11 Eco RV FIG. 1. Aligned nucleotide sequences of the visna virus (top), and CAEV (bottom) promoter regions (25). The numbers indicate nucleotide positions relative to the visna virus cap site. Homologous nucleotides are indicated with asterisks. The locations of AP-1-related sequences in the visna virus promoter or the CAEV promoter are indicated with arrows. The positions of TGCT sequences are indicated with unlabeled arrows. The positions of the TATA box and putative AP-4 sites are also indicated for each virus. A schematic of the visna virus LTR is shown below the aligned sequences. The U3, R, and U5 boundaries of the LTR and restriction sites used in the construction of mutations of the promoter are indicated. ylation of [14C]chloramphenicol (Dupont, NEN Research and the primer extension reaction was incubated at 37°C for Products) after various times (1 to 16 h) was measured by 15 min. After this incubation, 4 plI of 0.25 M EDTA-1.5 M separating the acetylated and unacetylated forms by thin- sodium acetate-75 pI of ethanol was added and the DNA layer chromatography, followed by liquid scintillation count- was precipitated. The DNA pellet was washed with 70% ing of spots cut from the plate. Preliminary experiments ethanol and then suspended in 10 pI of deionized H20. done by using these conditions indicated that the CAT assay Samples of this reaction were then run on 6% acrylamide-7 was linear with respect to enzyme concentration for up to 6 M urea sequencing gels (see Fig. 8a and b). h, provided that the percent acetylation of chloramphenicol To ensure that the differences between uninfected and was less than 60%. When incubations longer than 6 h were infected cells were not the result of differences in plasmid done, all samples to be compared (i.e., different mutants or DNA uptake or increased plasmid stabilization in infected infected versus uninfected extracts) were incubated for the cells (1), total cellular DNA was prepared from uninfected same length of time. All CAT assay data reported in this and infected GSM and SCP cells at 48 h after transfection paper are from points in the linear range of the assay, except and analyzed by Southern analysis. DNA was also prepared those noted otherwise. The results are expressed as the ratio from cells treated with DNase I before lysis and DNA of the percent conversion of [14C]chloramphenicol to its extraction to measure the amounts of DNA internalized by acetylated derivatives by a given mutant relative to the the cells. Most of the plasmid DNA was sensitive to DNase wild-type promoter (pVIS-LTR-CAT). Plasmids pSV2CAT, treatment in both GSM and SCP cells, indicating that most of pRSVCAT, and pTKCAT (pPOH3), used as controls in the plasmid DNA remained on the cell surface with the these experiments, have been previously reported (20, 21, DEAE-dextran transfection technique. No significant differ- 45). ences between the DNA contents of uninfected and infected Primer extension analysis. GSM cells grown in 100-mm- GSM cells were seen, although there was a 22-fold increase diameter dishes were infected with visna virus strain 1514 or in CAT activity in infected cells. Similar results were ob- mock infected. At 48 h later, the cells were transfected with tained in other experiments with both GSM and SCP cells. 3 ,ug of pVIS-LTR-CAT per ml in 200 ,ug of DEAE-dextran per ml of modified Eagle medium. At 48 h after transfection, RESULTS the cells were scraped into diluent, washed once with phosphate-buffered saline, and then solubilized in 4 M guani- Localization of transcriptional control elements in the visna dine thiocyanate-25 mM sodium citrate (pH 7.0)-0.1 M virus LTR by deletion analysis. The visna virus and caprine P-mercaptoethanol (8). RNA was purified by being pelleted arthritis-encephalitis virus (CAEV) LTRs are active in the through 5.7 M CsCI-0.1 M EDTA at 20°C at 35,000 rpm for same cell types both in tissue culture and in vivo (25; 16 h in a Beckman TLS 55 rotor. The pelleted RNA was unpublished data). This suggests that homologous regions suspended in deionized H20 and then desalted by reprecip- between the two LTRs are important for transcriptional itation with ethanol. For primer extension, 30 ,ug of total regulation. The LTRs of the two viruses are only about 50% cellular RNA was combined with 500,000 cpm of an approx- homologous overall; however, specific regions in the LTRs imately 265-bp uniformly labeled probe complementary to are highly conserved (Fig. 1). To identify the sequences in sequences in the 5' end of the cat gene, and the mixture was the visna virus LTR required for transcriptional activity, we dried. The dried RNA-primer mixture was suspended in 30 constructed a series of 12 5' (Fig. 2) and 11 3' (Fig. 3) pul of hybridization buffer (23), heated to 85°C for 10 min, and deletion mutants by using plasmid pVIS-LTR-CAT, which then allowed to anneal for 5 to 12 h at 37°C. The annealed contains the entire visna virus LTR driving transcription of primer-RNA mixture was precipitated with 600 ,u1 of etha- the cat gene (25), as well as an additional 6 5' deletion nol-3 M sodium acetate (2.5:1), and the pellet was washed mutants truncated at their 3' end at +4 (Fig. 2). These latter with 70% ethanol and then suspended in 30 p.l of reverse mutants permitted measurement of the activities of upstream transcriptase buffer (23). Avian myeloblastosis virus reverse promoter sequences in the absence of downstream viral transcriptase (20 U/pd; Pharmacia) was added (1 p,l), and the sequences present in the mRNA transcript. primer extension reaction was incubated at 22°C for 10 min The basal activities of these deletion mutants were mea- and then at 46°C for 30 min. Following these incubations, 1 sured in sheep alveolar macrophages and mouse L cells, in ,ul of RNase A (0.25 ,ug/,I; Calbiochem-Behring) was added which the basal activity of the visna virus LTR is high 3004 HESS ET AL. J. VIROL.

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(3 /+91) (3'/+70) (3/+57) (3'/+42) (3'/+29) (31/+21) (3'/+14) (3'/+4) (3'/-5) l (3 /-22 MAAM (3 /-36) IP (+23/+94) FIG. 3. 3' deletion mutants of the visna virus LTR. The top line is the nucleotide sequence of the downstream promoter and R regions of the visna virus LTR. The locations of the TATA box and TGCT sequences are indicated. Vertical lines indicate boundaries of sequences verified by dideoxy sequencing. The 5' endpoints of the mutants shown are at a HioidIll site at +452 that is upstream from the 5' border of the visna virus LTR. regardless of the serum concentration in the medium, and in SCP cells and GSM cells, in which the promoter is depen- dent on viral infection or high concentrations of serum (10%) for high levels of activity (for studies of the basal activity of the visna virus LTR, 10%c FBS was used for mouse L cells TABLE 1. Activities of 5' and 3' deletion mutants of the visna and GSM and SCP cells and 20%Y lamb serum was used for virus LTR in uninfected cells the macrophage cell line). % of wild-type promoter CAT activity in": Significance of the 43-bp repeats in the visna virus LTR. Mutant The activity of a given 5' deletion mutant was highly L cells Macrophages GSM cells SCP cells dependent on the cell type in which the plasmid was assayed pVIS5'( - 140) 80' 115" 149" 99 (Table 1: Fig. 4). The visna virus LTR contains a pair of pVIS5'(- 124) 47/" 96h 98 50 43-bp repeats located between -183 and -97 relative to the pVIS5'(-111) 48" 73" 57 24 cap site. Deletion of one 43-bp repeat (the -140 mutant) had pVIS5'( - 104) 57" 87" 78 16 relatively little effect on promoter activity in all of the cell pVlS5'(-99) 53^ 77 34 10 types. However, deletion of both repeats (the -99 mutant) pVIS5'(-91) 68" 61 39 10 reduced the activity of the promoter to less than 10% of that pVIS5'(-81) 25h 30 25 4.2 of the wild-type LTR in SCP cells and to 34% of wild-type pVIS5'(-67) 16 17 22 4.6 in Deletion of both had much pVIS5'(-61) 4.4 3.2 18 4.8 levels GSM cells. 43-bp repeats pVIS5'(-49) 5.9 0.8 2.5 4.6 less effect in the other cell types. The results obtained with pVIS5'(-37) 1.9 0.6 1.3 1.9 5' deletion mutants truncated at +4 were similar (Table 1, pVIS5'(-26) 4.3 0.8 1.4 1.9 bottom; see also Fig. 2). In this deletion series, deletion from pVIS3'(+91) 123' 135 184" 180" -140 (Table 1, mutant -140/+4; Fig. 2) to -107 (-107/+4) pVIS3'(+70) 153 114 228" 159" caused roughly a fivefold drop in the activity of SCP cells, a pVIS3'(+57) 56" 92 152" 113 threefold drop in GSM cells, a twofold drop in macrophages, pVIS3'(+42) 67 78 85 49 but no significant decrease in L cells. Additional decreases in pVIS3'(+29) 95 44 41 39 activity occurred in all of the cell types with continued pVIS3'(+21) 137 40 43 39 in SCP to pVIS3'(+ 14) 131 64 39 37 deletion to -94 (-94/+4). Thus, transcription and, pVIS3'(+4) 119 50 24" 35 a lesser extent, GSM cells is highly dependent on the pVIS3'(-5) 107 43 18" 16 sequences contained within the 43-bp repeats from -140 to pVIS3'(-22) 0.9 0.5 1.1 2.9 -99. pVIS3'(-36) 0.4 0.3 1.1 1.4 Deletional analysis of proximal promoter sequences. Dele- pVIS3'(- 140/+4) 177" 46 52" 28 tion of sequences from -91 to -81 caused roughly a 50% pVIS3'(- 107/+4) 171" 23 17'" 5.7 decrease in activity in all of the cell types tested (Table 1). pVIS3'(-94/+4) 73" 15 11i 2.2 This region contains the sequence TCTGCTTTT, which is pVIS3'(-83/+4) 118" 15 20" 3.3 4.1 8.5" 1.9 identical to a sequence in the noncoding strand of the visna pVIS3'(-65/+4) 85" +4 +12. A similar pVIS3'(-49/+4) 2.2" 0.3 1.0" 1.6 virus LTR located between and sequence. pVIS-LTR-CAT 100 100 100 1(0 CTGCTTTT, is found in the HIV LTR between -19 and -25 (63). Another deletion spanning this conserved element ' The values are averages of six independent transfections, except when in constructs -94/+4 and -83/+4 did not result in a drop in noted otherwise. L-cells. SPC cells, and GSM cells were grown in modified in this is Eagle medium containing 1OCi FBS. Macrophages were grown in Dulbecco activity, so that the significance of sequences region modified EaLgle medium containing 20% lamb serum. not clear. Average of 12 transfections. Continued deletion of 5' sequences from -81 down to -49 Average of 11 transfections. 3006 HESS ET AL. J. VIROL.

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FIG. 5. CAT assay of 3' deletion mutants. L cells, alveolar FIG. 4. CAT assays of 5' deletion mutants of the visna virus macrophages, and GSM and SCP cells were transfected with 3 ,ug of LTR. L cells, alveolar macrophages, and GSM and SCP cells were and cell the indicated 3' deletion mutant, and cell extracts were prepared for transfected with 3 jig of the indicated 5' deletion mutant, CAT assay 48 h later. Only the acetylated chloramphenicol product extracts were prepared for CAT assay 48 h later. Only the acetylated is shown for each mutant. These assays were from experiments chloramphenicol product is shown for each mutant. These assays summarized in Table 1. W.T., Wild type. were from experiments summarized in Table 1. severely reduced the activity of the visna virus LTR in all of virus LTR with linker-scanner mutants. The results of these the cell types, except in SCP cells, in which the activity of deletion experiments suggest that, in addition to the TATA deletions to -80 was already at a very low level (Table 1). box, the visna virus LTR contains multiple transcriptional This region between -81 and -49 spans a 13-bp sequence, control elements located between -140 and -49 relative to CAGCTGATGCTT, located at approximately -60, that is the cap site. To locate sequences which regulate transcrip- perfectly conserved between visna virus and CAEV. This tion within this region, a series of internal deletion and sequence is highly homologous to the recognition sequence linker-scanner mutations was constructed from libraries of 3' for transcriptional factor AP-4 ([T/C]CAGCTG[T/C]GG), and 5' deletion mutations of the visna virus LTR. Ligation of which activates simian virus 40 late transcription in vitro the appropriate deletion mutants created LTRs containing (40). This 13-bp conserved region is flanked on each side by clusters of point mutations in the region of viral DNA sequences homologous to the binding site for AP-1 (Fig. 2). replaced by an 8-bp KpnI linker. Twenty such internal Deletion from -81 to -67 moderately reduced activity in deletion or linker-scanner mutants were constructed, span- L cells and alveolar macrophages (roughly 40%) but had ning the region from -140 to +31 (Fig. 6). An additional little effect in GSM or SCP cells. Continued deletion to -61, internal deletion mutant, +23/+94, was made by deletion of which disrupted the putative AP-4 site, caused a nearly pVIS-LTR-CAT sequences between EcoRV and SstI (this fourfold drop in activity in L cells and a greater-than-fivefold construct does not contain an inserted KpnI linker [Fig. 3]). drop in macrophages. Further deletion to -49, which inter- The activities of these internal deletion and linker-scanner rupted the proximal AP-1-binding site, reduced activity mutants were tested in L cells, macrophages, and GSM and fourfold in macrophages and approximately sevenfold in SCP cells. GSM cells. This series of mutations identified two regions in the visna Role of transcribed sequences in the efficiency of gene virus LTR that were important for basal transcriptional expression-results of 3' deletion analysis. As with the 5' activity (Table 2). The first of these is the TATA box, which deletion mutants, the relative activities of the different 3' was important in all four cell types. Mutations affecting this deletion mutants also varied in different cell types (Table 1; sequence, -25/-17 and -22/-14, caused a large reduction in Fig. 5). Deletion of the U5 region of the LTR had little effect promoter activity in all of the cell types. The second impor- on promoter activity. The +91 and +70 3' deletion mutants tant region identified by the linker-scanner mutants was had higher activities than the wild-type promoter in all of the located between -71 and -49. This region contains the cell types tested. A roughly twofold drop in activity was seen putative AP-4-binding site flanked on both sides by AP- when additional sequences from +57 to +42 were deleted in 1-binding sites. In SCP cells, in particular, linker-scanner both GSM and SCP cells. The significance of this is not mutations in these sites significantly reduced activity (Table clear; however, the region that is present in mutant +57 and 2). Together with the deletion mutant data, these results absent in mutant +42 is highly G+C rich (12 of 15 bp are strongly suggest that the AP-1-related sequences and the G- C [Fig. 3]). putative AP-4-binding site in the region between -81 and The activities of mutants with 3' deletions continuing to -49 are important for the transcriptional activities of the +4 reduced the activity most in SCP and GSM cells, in visna virus LTR. which the +4 deletion had only 35 and 24%, respectively, of Localization of sequences required for activation of tran- wild-type activity. In contrast, the +4 deletion had greater- scription by serum. The activities of both the visna virus and than-wild-type activity in L cells and 50% of wild-type CAEV LTRs were strongly stimulated by high concentra- activity in transformed macrophages. In all of the cell types, tions of serum in the culture medium (Fig. 7a). To locate the deletions extending into the TATA box located between -22 serum-responsive sequences in the visna virus LTR, we and -15 reduced the activity of the LTR to barely detectable tested the activity of a subset of deletion mutants in cells levels. grown at either a high (10% FBS) or a low (0.5% FBS) serum Fine mapping of transcriptional control signals in the visna concentration. GSM and SCP cells were cultured in media VOL. 63, 1989 MUTATIONAL ANALYSIS OF THE VISNA VIRUS LTR 3007

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TABLE 2. Activities of internal deletion and linker-scanner TABLE 3. Stimulation of promoter activity by a mutants of the visna virus LTR in uninfected cells high serum concentration % of wild-type promoter CAT activity in": Fold increase in CAT activity in": Mutant L cells Macrophages GSM cells SCP cells Mutant GSM cells SCP cells -134/- 125 55" 1686 152 90 Expt 1 Expt 2 -128/-120 70 163 171b 128b -118/-92 104 148 104' 64 pVIS5'(- 140) 86 5.0 -118/-112 56 170 132d 102 PVIS5'(-91) 36 2.2 -90/-82 68 1356 181b 136b pVIS5'(-81) 70 4.1 -87/-81 74 128 171 76 pVIS5'(-67) 22 1.1 -71/-64 44 93 124" 67 PVIS5'(-61) 27 0.9 -67/-59 40" 127 158d 81 pVIS5'(-49) 2.7 1.1 -67/-50 42 136 81' 52 PVIS3'(+4) 31 2.5 -57/-50 44 108 75' 57 Col-TRE/TKCAT 1.2 4.2 1.2 -55/-44 47 102 59' 53 Col-TRE x 5/TKCAT 11.9 25 7.7 -40/-32 64 1116 156 1546 Col-TREA -72/TKCAT 1.0 1.4 1.3 -34/-27 157b 145 143" 79 pTKCAT (pPOH3) 1.5 -25/-17 63 12 12 8.7 pVIS-LTR-CAT 18.1 58 6.4 -22/-14 63 14 13 4.4 " The cells were grown in 10% FBS following transfection and compared - 10/- 1 57 124 194b 102 with cells grown in 0.5% FBS. The values reported for experiment 1 with -8/+ 1 101 139 101 227b GSM cells are averages of three independent transfections, while the values +51+1 101 98 83C 93 for the visna virus constructs used with GSM cells in experiment 2 and with +14/+27 128 86 84 60 SCP cells in experiment 1 are from single CAT assays done on pooled extracts +22/+30 128 84 121 137" from three separate transfections. The values for the Col-TRE constructs are +23/+94 144" 127 122 91 results of single transfections; however, similar results were obtained in other pCAEV-LTR-CAT 72 119b 61 99 experiments. pRSV-CAT 65 32b 12 7.4 pVIS-LTR-CAT 100 100 100 100 the 5' deletion mutants indicate that sequences upstream " The values are averages of six transfections, except when noted other- from -49 are required for serum responsiveness, while the wise. results obtained with the +4 3' deletion mutant show that b Average of 12 independent transfections. sequences downstream from the cap site were not respon- ' Average of nine independent transfections. d Average of 15 independent transfections. sive to serum. In particular, it appears that sequences located between -81 and -67 had positive effects on serum responsiveness in containing 0.5% FBS for 48 h before transfection. Following both GSM and SCP cells. No further decrease in serum transfection, the cells were grown in either 0.5 or 10% FBS responsiveness was seen in mutants with deletions between for 48 h before preparation of cell extracts for CAT assay. -67 and -61 in either cell type, indicating that the putative The results of these assays are summarized in Table 3, and a AP-4 site in the visna virus LTR is probably not involved in CAT assay of SCP cells grown at either a high or a low serum serum activation of promoter activity. The most dramatic concentration is shown in Fig. 7a. The results obtained with drop in serum responsiveness was seen with deletion of

a b -~SCP

wIGSM 0.5 1 0 0.5 10 0.5 1 0 0.5 1 0 0.5 1 0 0.5 10 W.T. -140 - 91 - 67 -49 +4

9h ~ ~ ... 0.5 10 0.5 10 0.5 10 'TRExl TREx5 TREx..\72 TK CAI TK CAT -K CAT FIG. 7. (a) Responsiveness of the visna virus LTR to serum stimulation. CAT assays were performed on extracts from SCP cells grown in either 0.5 or 10% FBS following transfection with 3 p.g of the indicated mutant. The percent conversions for this assay are shown in Table 3 (experiment 2). W.T., Wild type. (b) Responsiveness of plasmids containing synthetic AP-1 sites to serum. CAT assays were performed on extracts from GSM or SCP cells grown in either 0.5 or 10% FBS following transfection with 3 [±g of the indicated plasmid. The results of this assay are summarized in Table 3. VOL. 63, 1989 MUTATIONAL ANALYSIS OF THE VISNA VIRUS LTR 3009 sequences between -61 and -49. This sequence spans the extension reaction done on visna virus-infected GSM and proximal AP-1 site that perfectly matches the consensus SCP cells (Fig. 8a and b). In contrast, no initiation was sequence. Deletion of these sequences resulted in a tenfold detected in uninfected cells, except in one experiment using reduction in the response of GSM cells to serum. The serum RNA from GSM cells grown in 10% FBS (Fig. 8a). In this response was not detectable in SCP cells with deletions experiment, there was an approximately 51-fold increase in extending beyond -81. CAT activity and a minimum of a 31-fold increase in CAT To determine whether AP-1 sites alone could confer serum mRNA as estimated by laser densitometric scanning. While responsiveness, we tested the abilities of synthetic copies of the undetectable level of the extension product in uninfected the sequence TGAGTCA to stimulate the activity of the cells made quantitation of the fold increase in RNA in the herpes simplex virus tk promoter (2). Plasmids containing other experiments impossible, the increases in CAT RNA one copy (Col-TRE/TKCAT), five copies (Col-TRE x 5/ appear to account for at least half of the increase in CAT TKCAT), or a single copy (Col-TREA -72/TKCAT) of the activity observed in infected cells (a 57-fold increase in GSM mutant sequence GGAGTCA (obtained from M. Karin [2]) cells and a 116-fold increase in SCP cells in the experiments were transfected into GSM and SCP cells which were then shown in Fig. 8b). Thus, it appears that visna virus trans- grown at either a high or a low serum concentration as activation is mediated at one level by increases in the described above. steady-state concentration of mRNA. The results of our experiments with the constructs con- Deletional analysis of sequences required for trans-activa- taining synthetic AP-1 sites (Fig. 7b) indicate that these tion. To determine which LTR sequences are required for sequences are responsive to serum in both GSM and SCP trans-activation, these sets of 5' and 3' deletions mutants cells. The activity of the Col-TRE-TKCAT construct re- were transfected into uninfected and visna virus-infected sponded approximately 4-fold at a high serum concentration, GSM and SCP cells and cells were harvested and assayed for while Col-TRE x S/TKCAT was activated 25-fold by a high CAT activity 48 h later. The results of these deletion mutant serum concentration in GSM cells. In contrast, mutant experiments are summarized in Table 4, and the results of a Col-TREA -72/TKCAT was not significantly activated by typical CAT assay of uninfected and visna virus-infected serum nor was the tk promoter (pTKCAT [pPOH3]; 45) GSM cells (cultivated at a low serum concentration) are alone. Similar results were obtained in other experiments shown in Fig. 9. with SCP cells; however, the activities of these constructs The results of experiments done with the 3' deletions were lower when Col-TRE x 5/TKCAT was activated indicate that the visna virus LTR responds to trans-activa- 7.7-fold. No activation was seen with the other two con- tion provided sequences upstream from -5 are intact (Table structs. Thus, it appears that multiple copies of the AP-1 4). However, in all of the 3' deletion mutants assayed in sequence increased responsiveness to serum in both cell GSM cells (at both low and high serum concentrations), the types. Taken together, the deletion mutagenesis experiments level of trans-activation was reduced by 50%. This suggests and the experiments with the AP-1-tk hybrid promoters that the 3' region contributes to the full trans-activation support the idea that the serum responsiveness of the visna observed. Curiously, the -5 3' deletion mutant (Table 1, virus promoter is mediated by AP-1-related sequences GSM cells, 10% FBS) had the highest fold trans-activation of present within the LTR. all of the deletion mutants (33-fold versus 24-fold for the trans-Activation by visna virus infection is mediated primar- wild-type LTR). Similar results were obtained with SCP ily by increasing steady-state levels of mRNA. To determine cells, in which the -5 deletion mutant was trans-activated whether trans-activation by visna virus infection is the result 10-fold compared with 4.4-fold for the wild-type promoter of increased levels of mRNA, the steady-state levels of CAT (trans-activation of the wild-type promoter and also that of mRNA transcribed by the visna virus LTR were measured in the -67 mutant were anomalously low in this experiment). uninfected and visna virus-infected GSM and SCP cells by In both cell types, continued deletion to -22, which deleted primer extension. GSM and SCP cells either infected with the TATA box, totally abolished both promoter activities visna virus (multiplicity of infection, 1) or mock infected and eliminated any detectable response to trans-activation. were transfected 48 h later with plasmid pVIS-LTR-CAT. One unusual finding was that the +4 deletion mutant and This plasmid contains the entire visna virus LTR driving the 5' deletion mutants truncated at +4 were all trans- transcription of the cat gene (Fig. 1). Total cellular RNA was activated poorly compared with similar deletion mutants prepared 48 h after the transfections. Depending on the truncated at +13 and -5. This discrepancy in trans-activa- experiment, the activity of the visna virus LTR (measured tion was seen only in cells grown at a high serum concen- by CAT assay) was increased by as much as 150-fold in SCP tration. Similar results were obtained in several other exper- cells and 320-fold in GSM cells by visna virus infection. iments. In addition, we found that a series of 5' deletion Plasmid DNA uptake was found to be equivalent for infected mutants truncated at +4 (see below) were all very poorly and uninfected cells (see Materials and Methods). To quan- trans-activated at a high serum concentration, although they titate the levels of CAT mRNA, 30 ,ug of RNA from were trans-activated at a low serum concentration. The uninfected and visna virus-infected cells was analyzed by reason why these mutants have such markedly lower activ- primer extension with a 265-bp uniformly labeled primer ities in visna virus-infected cells is not clear, but their homologous to 5' cat sequences. Ten pairs of primer exten- behavior suggests that sequences immediately adjacent to sion reactions were done on RNAs from five separate the initiation site are recognized by a trans-acting factor in transfections of uninfected and visna virus-infected GSM infected cells. cells (three experiments were done with GSM cells grown in The 5' deletion data indicate that the visna virus LTR 0.5% serum, and two experiments were done with GSM cells responds to trans-activation if5' LTR sequences out to -67 grown in 10% serum). Two additional pairs of primer exten- are intact (Table 4). In an experiment done with GSM cells sion reactions were done on RNA from SCP cells grown in (2% FBS), the -67 deletion mutant was trans-activated 0.5% serum that were transfected with pVIS-LTR-CAT. 60-fold compared with 78-fold for the wild-type LTR. In this High levels of the primer extension product of the correct same experiment, further deletions into the promoter de- length (approximately 549 bp) were found in every primer creased the trans-activation effect to roughly 15-fold for the 3010 HESS ET AL. J. VIROL.

a 1 2 3 4 b SCP

622 *.... 527

GSMv4

404 - - _

P:.2

- E - _ sCN ;i C T

FIG. 8. (a) Typical primer extension on uninfected and infected GSM cells. Primer extension reactions were performed on 30 p.g of total cellular RNA from uninfected and visna virus-infected GSM cells grown in 0.5% FBS (lanes 1 and 2, respectively) or on uninfected and visna virus-infected GSM cells grown in 10% FBS (lanes 3 and 4, respectively). The positions of the primer extension products are indicated. The marker lane is an HpaII digest of pBR322 that was end labeled with polynucleotide kinase. The numbers to the left indicate molecular sizes in nucleotides. (b) Primer extensions and laser densitometric scans of transfected GSM and SCP cells. Overexposed films of primer extension products from uninfected and visna virus-infected SCP and GSM cells are shown. Densitometric scans of shorter exposures of the same films are shown to the right; these were done with an LKB soft laser densitometer.

-61 mutant and 2-fold for the -49 mutant. Unfortunately, bles 5 (GSM cells) and 6 (SCP cells). None of the linker- the interpretation of these results is hampered by the low scanner or internal deletion mutants abolished the response basal activities of the latter two mutants in uninfected cells. to trans-activation. Mutations affecting the TATA box (mu- This low basal activity tends to cause underestimation of the tants -25/-17 and -22/-14) had low levels of activity in trans-activation effect. both uninfected and infected GSM and SCP cells. These data To obviate this difficulty, other experiments were done in indicate that although the TATA box is an important tran- GSM cells grown in 10% FBS, which raised the basal level of scriptional regulatory element, it is unlikely to be the target promoter activity so that it was accurately measured (these for trans-activation. results are summarized in column 2 of Table 4). As was Mutations in the proximal promoter region between -67 observed under low-serum conditions, the 5' deletion mu- and -44 impaired the response to trans-activation. This tants between -140 and -67 responded to trans-activation region contains a putative binding site for AP-4 and a as well as did the wild-type LTR (although their overall level sequence identical to the consensus recognition sequence for of activity was lower). The -61 deletion mutant was also AP-1 located just downstream from this site. The -67/-59 trans-activated well (21-fold), while continued deletion to mutant disrupts a putative AP-4-binding site that is con- -49 reduced the effect moderately (15-fold). trans-Activa- served between CAEV and visna virus. This mutant had tion was observed even with the -26 mutant, which contains essentially wild-type activity in infected GSM and SCP cells. only the TATA box and downstream sequences. In SCP Since there is only one copy of the putative AP-4 sequence cells, trans-activation was observed for 5' deletions extend- in the visna virus LTR, this suggests that this putative AP-4 ing as far as -61. Beyond this point, the basal activities of site is not the target for trans-activation in visna virus- the mutants were extremely low and no significant trans- infected cells. The -67/-50 mutant, which differs from the activation was observed. Thus, the results obtained with our -67/-59 mutant by lack of the 9-bp sequence TGCTTG 5' deletion mutants suggested that the sequences between AGT, had dramatically reduced activity. This region con- -67 and -49 are involved in the trans-activation process. tains the sequence TGCTT, which is found in the CAEV This region spans a sequence that is identical to the consen- LTR in the same location (Fig. 1). This TGCTT sequence is sus recognition sequence for AP-1. also repeated multiple times in the visna virus LTR, includ- To locate regulatory sequences within the region that the ing a copy between -90 and -86. The sequences common deletion mutants identified as essential for basal activity and between the 9-bp sequence described above and the se- for trans-activation, the internal deletion and linker-scanner quences mutated in the -57/-50 and -55/-44 mutants are mutants were transfected into uninfected and visna virus- the five nucleotides TGAGT. In the noncoding strand, these infected SCP and GSM cells, and CAT activity in cell are the nucleotides ACTCA, which form an essential part of extracts was determined 48 h later. The results of these the recognition sequence for AP-1 located between -54 and linker-scanner mutant experiments are summarized in Ta- -48 (TGACTCA). VOL. 63. 1989 MUTATIONAL ANALYSIS OF THE VISNA VIRUS LTR 3011

TABLE 4. Responses of 5' and 3' deletion mutants of the visna TABLE 5. Responses of internal deletion mutants and virus LTR to trans-activation linker-scanner mutants of the visna virus LTR to tratns-activation in GSM cells" Fold increase in CAT activity in': % Wild-type activity Fold tranis- Mutant GSM cells SCP cells Mutant activation 2'% FBS 10% FBS (10% FBS) 2% FBS 10% FBS 2% FBS 10% FBS pVIS5'(- 140) 115 18 76 8 -134/-125 181 215 46 pVIS5'(- 124) 50 22 107 10 -128/-120 125 180 31 pVIS5'(-111) 64 23 37 6.7 -118/-92 116" 104 28 5.6 -118/-112 93 115 19 pVIS5'(- 104) 43 22 45 27 pVIS5'(-99) 45 34 50 4.0 -90/-82 80 180 pVIS5'(-91) 128 23 38 7.0 -87/-81 124 124 23 pVIS5'(-81) 45 31 24 11 -71/-64 100 90 20 60 23 11 1.7 -67/-59 130 168 21 pVIS5'(-67) -67/-50 39 89 30 pVIS5'(-61) 15 21 3.2 4.1 31 pVIS5'(-49) 2.1 15 3.1 0.9 -57/-50 15 85 14 1.4 NA -55/-44 21 62 29 pVIS5'(-37) 1.3 -40/-32 64 151 31 pVIS5'(-26) NA" 15 NA NA 37 pVIS3'(+91) 50 8 57 6.9 -34/-27 109 210 7.1 -25/-17 13 11 28 pVIS3'(+70) 9 11 10 23 pVIS3'(+57) 11 7.1 -22/-14 - 10/- 1 103 179 24 pVIS3'(+42) 17 23 99 31 pVIS3'(+29) 15 17 -8/+1 104 24 +5/+1 103 62 22 pVIS3'(+21) 39 13 77 +14/+27 109 174 67 pVIS3'(+ 14) 15 16 71 19 pVIS3'(+4) 3.4 1.3 +22/+30 60 10 +23/+94 167" 199 53 pVIS3'(-5) 24 33 42 pVIS-LTR-CAT 100 100 32 pVIS3'(-22) NA NA NA NA pCAEV-LTR-CAT 50 pVIS3'(-140/+4) 3.4 0.6 2.2 pVIS3'(-107/+4) 1.5 0.6 pRSV-CAT pVIS3'(-94/+4) 1.0 0.9 " The values reported for cells grown in 0.5% FBS are averages of triplicate pVIS3'(-83/+4) 1.5 0.7 trnasfections. while the values reported for GSM cells grown in 2 and 10% pVIS3'(-65/+4) 2 NA FBS are averages of six independent transfections, except as noted. pVIS3'(-49/+4) NA NA " Average of three transfections. pVIS-LTR-CAT 78 24 66 4.4 visna virus-Intectea cells were compared{ z: ..witn_. :uninte:_**1...... 11c.eu eiswFA_. ,;1inc ;t ...... ,1 percent conversions for the wild type and -140 and -124 5' Jeletion mutants Copies of a synthetic AP-1 site confer responsiveness to viral in SCP cells (2% FBS) were beyond the linear range of the CAkT assay, so that trans-activation on a heterologous promoter. The results of the fold trans-activation may have been underestimated for these constructs. the deletion and linker-scanner mutant experiments suggest The values for the GSM and SCP cells represent averages of ttwo independent that the for visna virus trans-activation are transfections for cells grown in 2% FBS, except for pV target sequences pVIS5'(-124) in SCP cells and pVIS3'(+21) in GSM cells, vvhich are results not distinct from the binding sites for cellular transcriptional of single transfections. The results for cells grown in 10% FBSSare averages of factors (i.e., trans-activation cannot be totally eliminated six transfections. without abolishing the basal activity of the promoter as " NA, Negligible CAT activity. well). Mutations that impaired trans-activation were located in regions upstream of the promoter that contain putative The other region in which mutations imp)aired trcans- AP-1-binding sites. To test the hypothesis that trans-activa- activation was located within the 43-bp repezats. Mutants tion is mediated via AP-1 sites, we tested the abilities of -118/-92 and -118/-112 had significantly lo)wer activity synthetic copies of the sequence TGAGTCA to stimulate the and trans-activation in visna virus-infected SCP cells. In activity of the herpes simplex virus tk gene by using plasmids low-serum medium, these mutants also had reduced activity generously provided by M. Karin (2). The transcriptional in visna virus-infected GSM cells. Interestingly, these mu- activity of the tk promoter depends on the binding of two tants overlap the sequence TGACACA, whicih is homolo- transcriptional factors, SP1 and CTF, whose recognition gous to the AP-1 recognition sequence and has been shown sequences are not present in the visna virus LTR (for a to bind AP-1 in vitro (40). review, see reference 38). Plasmids containing no copies of the AP-1 sequence (pTK-CAT) (45), one copy (Col-TRE/ TKCAT) or five copies (Col-TRE x 5/TKCAT) of the AP-1 Ot ^ recognition sequence TGACTCA, or a single copy of the I . I mutant sequence TGACTCC (Col-TREA -72/TKCAT) (2) were transfected into uninfected or visna virus-infected w--vwb GSM or SCP cells, and the cells were harvested for CAT iVt V assay 48 h later. The results of these experiments (Table 7) indicate that the AP-1-containing plasmids were responsive iI il Ail rAli f. P'l fvi l .- c -91 617 - W.T. to tratns-activation in both GSM and SCP cells (Fig. 10). FIG. 9. Responsiveness of the visna virus LTR to viral trans- activation in GSM cells. CAT assays were performed on extracts of DISCUSSION uninfected and visna virus-infected GSM cells growln in 0.5% FBS and transfected with 3 p.g of the indicated plasmid. W .T., Wild type, The results of our deletion and linker-scanner mutation M, mock infection. experiments with uninfected cells suggest that different 3012 HESS ET AL. J. VIROL.

TABLE 6. Responses of internal deletion mutants alnd (SM sCp MOGK linker-scanner mutants of the visna virus LTR to trans-activation in SCP cells S % Wild-type activity Fold trails- MutantMutant a~~~~~~~~~~~~~~~ctivation 0.5%7 FBS 2% FBS 10% FBS 0.5%1 FBS 9 v.v9v.999.*0 v*

I, -134/-125 119 88 163 207 ; C l: .\,r TED -128/-120 89 92 121 106 -118/-92 31 28 29 38 -118/-112 39 47 48 66 -90/-82 105 115 131 116 -87/-81 141 120 157 87 -71/-64 76 59 92 118 -67/-59 70 96 93 143 -67/-50 7.6 14 12 36 -57/-50 13 24" 10 62 -55/-44 12 14 12 59 FIG. 10. Response of plasmids containing synthetic AP-1 to viral -40/-32 36 82 70 85 tr(ans-activation. CAT assays on extracts from uninfected or visna -34/-27 72 94 172 126 virus-infected GSM or SCP cells grown in either 0.5% (SCP) or 10% -25/-17 12 8.7 9.8 96 (GSM) FBS following transfection with 3 ,ug of the indicated -22/-14 18 9.7 14 91 plasmid. RSV, Rous sarcoma virus; SV40, simian virus 40. - 10/- 1 71 103 122 129 -8/+1 130 99 71 158 +5/+1 77 66 26 153 by using a combination of deletion and linker-scanner mu- +14/+27 80 69 79 142 tants. We identified three regions in the visna virus U3 +22/+30 63 61 70 132 region that are important for transcriptional regulation. +23/+94 91' 170 These regions include the TATA box and the proximal pVIS-LTR-CAT 100 100 100 155 promoter region between -81 and -49, which were impor- tant in all of the cell and the direct " The values reported for cells grown in 0.5% FBS ar-e averages of triplicate types tested, 43-bp transfections, while the values reported for GSM cells grown in 2% FBS are repeats located farther upstream, which were essential in averages of six independent transfections. except as noted. The values some cell types but not in others. The latter two regions each reported for cells grown in 10% FBS are averages of nine independent contain two copies of an AP-1-like sequence. In addition, the transfections. a " Average of five transfections. proximal promoter region contains sequence homologous Average of three transfections. to the recognition sequence for AP-4 (40). The 43-bp repeat located between -140 and -97 was essential for high activity in SCP cells and was somewhat regions of the visna virus LTR are important for transcrip- important in GSM cells but was less important in the other tional activity in different cell types. The visna virus LTR is cell types. This region contains two putative AP-1-binding highly redundant, with six copies of sequences related to the sites. One is located in the noncoding strand between -127 binding site for AP-1, as well as other upstream sequences and -121, and the other is located further downstream in the that appear to have arisen from duplication of proximal 43-bp repeats (-116 to -111), and deletion of this region promoter sequences. Such redundancies make it difficult to (-124 to -111) resulted in substantial reduction of activity in locate specific sequences required for transcriptional activity GSM (40%) and SCP (50%) cells. This latter sequence is by linker-scanner mutational analysis, because mutations in homologous to the sequence TGACAAA, which is found in one site can be compensated for by another copy of the the same location in the 71-bp repeats of CAEV, and is control element at another site. Such sites can be identified identical to a sequence in the simian virus 40 promoter that has been shown to bind purified AP-1 (40). Deletion analysis identified a proximal promoter region TABLE 7. Responses of constructs containing synthetic AP-1 located between -81 and -49 that was necessary for pro- sites to viral trans-activation moter activity in all of the cell types tested. This region contains a sequence homologous to transcriptional factor Fold increase in CAT activity in": AP-4, which is immediately adjacent to the AP-1-TGCT Construct GSM cells SCP sequence. This sequence element, which is conserved be- tween CAEV and visna virus, is flanked in both viruses by a Expt 1 Expt 2 cells pair of putative AP-1 sites. All AP-4 sites identified have Col-TRE/TKCAT 2.3 29 7.2 been found in close association with AP-1 sites, and AP-1 Col-TRE x 5/TKCAT 36 23 20( and AP-4 have been shown to cooperatively activate tran- Col-TREA -72/TKCAT 7.4 11 1.9 scription in vitro (40). The distal AP-1 site (located between pTKCAT(pPOH3) 0.6 -77 and -71) appears to be important in mouse L cells and pVIS-LTR-CAT 14 12 >23 macrophages, while the proximal AP-1 site appears to be pRSV-CAT 4.0) 1.5 in all The pSV2CAT 1.7 21 important four cell types. sequences homologous to the AP-4 site were shown to be important by the 5' " Visna virus-infected cells wer-e compair-ed with Lininfected cells. Expeli- deletion experiments with both macrophages and mouse L ments 1 and 2 with GSM cells were done with 10%; FBS. The values r-epor-ted cells, and we noted that deletion of the putative AP-1 site are the results of single tr-ansfections for experinient 1 and triplicatte tr-ansfec- tions for experiment 2. The experiment with SCP cells was done with medium between -77 and -71 upstream from the AP-4 sequence had containing 0.5% FBS. and the results of this experinient arc averages of a more deleterious effect in cell types (macrophages and L duplicate transfections. cells) in which the AP-4 site was important. This suggests VOL. 63. 1989 MUTATIONAL ANALYSIS OF THE VISNA VIRUS LTR 3013 that, as has been described for simian virus 40 (40), the AP-4 be analogous to one of those of the DNA viruses, which have site and the upstream AP-1 site bind transcriptional factors been shown to encode trans-activator proteins that increase that synergistically activate transcription. transcription from specific viral promoters (for reviews, see The deletion mutants tested in cells grown in both high- references 4, 28, and 38). Of these viral trani.s-activators, and low-serum media strongly implicate the multiple copies many have been shown to indirectly influence promoter of AP-1 sites, rather than the putative AP-4 site, as the activity by increasing the binding of cellular transcriptional sequences through which the response to serum is mediated. factors to viral promoter sequences. The adenovirus ElA It is likely that having multiple copies of AP-1-like sequences protein, for example, activates transcription from numerous in lentivirus LTRs (six copies in visna virus, five copies in different adenovirus early promoters that have little se- CAEV; Fig. 1) enhances the activation of these promoters quence homology. The protein appears to increase the by serum, since our experiments with synthetic AP-1 sites in binding factor of transcriptional factor E2F to the ElA and hybrid promoter constructs indicated that five copies of the E2A promoters (31, 32, 58). In some other promoters, ElA AP-1 sequence responded sixfold more to serum than did a increases the activity of a factor that interacts with the construct containing a single AP-1 site. In addition, the TATA box (35, 68). presence of multiple copies of this transcriptional control The data presented here suggest that the transcriptional element would be expected to have a strong evolutionary component of visna virus tratn.s-activation is mediated via advantage. Mutations arising in one site could be compen- similar interactions between viral trans-acting factors and sated for by other sites in the promoter. cellular factors that recognize AP-1 sites in the viral LTR. The proximal AP-1 site and the sequences that flank it There are multiple copies of the AP-1 sequence in similar show an interesting homology with both CAEV and the locations in both the visna virus and CAEV LTRs. Both enhancer region of c-fos, which is another serum-responsive LTRs are strongly responsive to trans-activation. The dele- promoter (Fig. 1; for a review offo.s regulation, see reference tion mutant experiments reported here that were done with 65). In each promoter, there is an upstream TGCT sequence, low-serum medium showed loss of trans-activation (as well followed by an AP-1-like sequence, and the sequence CAG as basal promoter activity) when the last remaining AP-1 site ATGT. The visna virus promoter contains a 9-bp sequence, in the promoter was deleted. The linker-scanner mutants CGCAGATGT, that is identical to a sequence in the fo.s that interrupted the proximal AP-1 site and, to a lesser enhancer. Interestingly, this sequence overlaps part of the extent, the AP-1-related sequence between -118 and -111 dyad symmetry element that constitutes the serum-respon- impaired tr-tiis-activation in SCP cells. Finally, in this report sive element of c-lo.s (48, 64, 65). This region of the visna we show that synthetic copies of the AP-1 sequence ren- virus promoter is also partly homologous to a sequence from dered the herpes simplex virus tk promoter, a promoter the promoter region of equine infectious anemia virus, whose activity is not stimulated by visna virus infection, another macrophage-tropic lentivirus (12). responsive to viral tran.s-activation. These studies of visna virus tran.s-activation demonstrate The deletion and linker-scanner mutations described here that the steady-state level of viral mRNA is increased in provide genetic evidence that AP-1 plays a pivotal role in virus-infected cells. The increase in RNA, however, does both the developmental regulation of visna virus gene not account for the entire increase in tr-a,i.s-activation ob- expression and the stimulation of the viral promoter by served. This suggests that tranzs-activation of visna virus is virally encoded trani.s-acting factors. One important area of mediated at multiple levels, one being the increase in the investigation will be to determine whether any cellular genes steady-state level of RNA. The genetic analysis of the are activated by visna virus infection. If visna virus tranl.s- (is-acting sequences responsive to the viral tranits-activation activation is mediated via AP-1 sites, this suggests that visna supports this hypothesis. Sequences upstream (-61 to -49) virus replication could activate other cellular genes contain- of the viral promoter were found to be important (in low- ing AP-1 sites, such as collagenase (2) or c:ftos (47). One serum medium) for high levels of trans-activation. However. would expect that activation of the latter in macrophages sequences 3' of the promoter which are transcribed in the might lead to prolonged and inappropriate macrophage acti- viral mRNA were found to contribute to tri'ans-activation in vation (26) that would give rise to the immunopathologic GSM cells and in SCP cells in low-serum medium. These changes seen in animals chronically infected with visna data suggest that trans-activation of visna virus, like that of virus. HIV or equine infectious anemia virus, is mediated by a bimodal mechanism. However, in visna virus no trans-acting ACKNOWVLEDGMENTS responsive element was identified in the 3' R sequences. We thank M. Karin and I). Hayward for gifts of plasmids. 0. Sequences downstream from -5 in the R region may be Narayan for the macrophage cell line, and V. Proctor for assistance involved in tr-anis-activation; however, levels of approxi- with tissue culture. mately 50% of wild-type trans-activation could be observed This work was supported by Public Health Service grants NS when these sequences were deleted. These results contrast 21916. NS 23039, and NS 16145 from the National Institutes of markedly with the organization of the HIV and equine Health and a grant from the National Multiple Sclerosis Society (RG infectious anemia virus promoters, in which sequences 1985-A-4). J.L.H. is supported by the National Institutes of Health present in the R region appear to be the primary target for Medical Scientist Training Program. J.A.S. is a fellow of the trans-activation (13, 52, 55). 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