Identification of sequences in the long terminal repeat of avian sarcoma virus required for efficient transcription

VIROLOGY

162,243-247

(1988)

Identification

of Sequences in the Long Terminal Repeat of Avian Sarcoma Virus Required for Efficient Transcription

SIDDARAME GOWDA,’ Department

of Microbiology,

AREPALLI S. RAO, YONG WOONG KIM,* AND RAMAREDDY V. GUNTAKA~ School Received

of Medicine, June

University

10, 1987;

of Missouri-Columbia,

accepted

September

Columbia,

Missouri

652 12

8, 1987

Two different vectors, LTR-NEO-LTR and LTR-CAT-LTR, were constructed and deletions were introduced in the upstream LTR at -299 and at -140. These deletion mutants were introduced into QT6 cells under transient expression conditions, and the levels of transcription were monitored by dot blot hybridization or by CAT assays. The results indicate that the nucleotides between -208 and -201 and between -141 and -119 in the U3 region of RSV LTR are Q 1988 Academic Press, Inc. required for efficient trSnSCriptiOn.

Long terminal repeats (LTRs), sequences of 300 to 1200 bp in length, are present in all retroviruses at the ends of the viral genome (22). They are required for viral DNA integration (18) and possess signals for transcription initiation and termination (22). The LTRs contain upstream regulatory domains that act as enhancers (3, 4, 11, 12, 15, 23). By virtue of these regulatory sequences they can activate adjacent cellular oncogenes, thus promoting oncogenesis (7, 19, 23). Luciw et a/. (12) and we (15) showed originally that the upstream sequences between nucleotides -300 and -55 are required for efficient transcription of linked genes. Recent analysis by other groups has confirmed these results and further identified two well-defined but spatially separated domains that are required for enhanced expression of genes (10). Here we provide evidence for the presence of different motifs that are required for efficient promotion of transcription. The sequences between nucleotides - 141 and -119 and between nucleotides -208 and -201 together or separately form the enhancer domain. We constructed a vector which carries a neomycin gene of bacterial transposon 5 (Tn5) under the control of the LTR of Prague C strain of Rous sarcoma virus (16). The neoR gene is flanked by two LTR sequences, and transcription initiation and termination take place in the upstream and downstream LTRs, respectively (15). The neoR gene, driven by the LTR, has been shown to be active in a variety of eukaryotic cells as well as in Escherichia co/i (15, 16).

We have introduced various deletions in the upstream LTR of this clone (PAN-6D3A) in the following way. pATV-6.9 (16) which is neoR and tep was completely digested with Sphl (Fig. 1A), and the fragments were ligated and introduced into E. co/i. Colonies which are ampR and te? as a result of the loss of the small Sphl fragment were selected. From one of these clones (Fig. lc), which has a single EcoRl site of -299, DNA was isolated, linearized with EcoRI, and digested with Ba131 nuclease. Following filling-in the cohesive ends with the large fragment (Klenow) of E. co/i DNA polymerase I, they were ligated and used to transform E. co/i. Transformants were selected on ampicillin plates, DNA was isolated from individual colonies, and the extent of deletions was determined. Details of these strategies as well as introduction of the neoR gene into various deletion mutants were as described in the legend to Fig. 1. pATV-CG-CAT clones were constructed by replacing the neo gene with CAT gene from Tn9, which was obtained as a BarnHI cartridge (Pharmacia, NJ). The maps of the final pATV-C6 NE0 and pATVC6 CAT clones are shown in Fig. 2. Ba131 deletions at the Sphl site (-141) in the upstream LTR were constructed and characterized as described in the legend to Fig. 1. The 250-bp EcoRl fragment (-54 to -299) of pATV-6D3A was subcloned in M 13mp7 (14) and Ba/3 1 deletions were made at the Sphl site in the LTR. The extent of deletion in each clone was examined by dideoxynucleotide sequencing (21). The 250-bp EcoRl fragment with appropriate deletion was then transferred to pATV-6D3AA250 ARI clone (Fig. 1D) in which the 250-bp EcoRI fragment had been deleted, and supercoiled DNAs were prepared from appropriate clones. Transfections were carried out in QT6 (quail) cells with 10 pg supercoiled DNA per loo-mm plate as de-

’ Present address: Tobacco and Health Research Institute, University of Kentucky, Cooper and Alumni Drive, Lexington, KY 40606. ’ Present address: Department of Agricultural Chemistry, College of Agriculture, Chonnam National University, Kwangju, Korea. ’ To whom requests for reprints should be addressed. 243

0042-6822/88 Copyright All rights

$3.00

0 1988 by Academic Press, Inc. of reproductron I” any form reserved.

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FIG. 1. Construction of vectors with various deletions. pATV-6.9, which has a LTR sequence from -299 to +44 (16) was digested with Sphl and the resulting large fragment (6) was ligated and used to transform E. co/i HBl 01. A transformant containing the LTR sequence from -299 to -141 (C) was selected and supercoiled DNA was prepared. It was opened up at the single fcoRl site at -299 and digested with Bal31 (13). Following filling-in with the Klenow fragment of DNA polymerase and ligation, it was introduced in f. co/i HBlOl Appropriate clones were selected and the extent of deletions was determined by sequencing or by Sl mapping (D). The 1.8-kb Sphl fragment from pATV-6D3A (E) containing neomycin gene was introduced at the single Sphl site of each deletion mutant (D). Transformants were selected on LB + neomycin plates (F). In order to introduce the U5 sequences in the downstream LTR, the Pstl-BarnHI fragment of pATV-6D3 (G) containing the origin of replication was isolated and ligated to the BarnHI-fstl fragment containing the neomycin gene(F) and the ligated DNA was used to transform f. co/i HBl 01. Transformants (H) with appropriate 5’deletions in the upstream LTR were selected on neomycin plates. For construction of deletion mutants of the Sphl (- 141) site, the fcoRl245-bp fragment (-299 to -54) in PAW-6.9 (A) was cloned in M 13mp7 (14). Form I DNA was isolated from appropriate clones, opened at the single Sphl site, and digested with Ba131 under appropriate conditions. The DNA was filled-in, ligated, and used to transform E. co/i JM 103. Single-stranded DNA was isolated from selected recombinants and the deletions were determined by sequencing (21). The EcoRl fragments (less than 245 bp) were purified from appropriate clones and transferred to pATV-6D3A A 250 (D). This clone is identical to pAW6D3A except it lacks the 245-bp fcoRl fragment in the upstream LTR.

scribed (15) using the calcium phosphate method (5). Various dilutions of RNA, ranging from 10 to 0.1 pg, were spotted on a nylon filter paper, and the filters were baked for 2 to 3 hr in vacua and hybridized for 40 to 44 hr with neomycin gene probe as described in the legend to Fig. 3 using the nick-translated neomycinspecific 0.7-kb Pvull fragment as the probe. In some experiments, the RNA was treated with ribonuclease-

free deoxyribonuclease, prior to hybridization. Following hybridization, the filters were exposed for appropriate periods at -70”. Each spot was then cut out and counted in a scintillation counter. The spots containing the normal XC or QT6 DNA were used as controls to substract the background levels of radioactivity. The amount of neomycin-specific RNA in pATV-C6 was taken as 1OOOb.The results of some clones with

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FIG. 2. Maps of the NE0 and CAT vectors. pATV-C6 NE0 vectors with various deletions at -299 (indicated by an arrow) were constructed as outlined in Fig. 1. pATV-6D3 CAT was constructed by introducing the SamHI CAT fragment at the SamHI site in pATV-6D3. Purified pAlV-6D3 CAT was digested with Sphl and the fragment containing the CAT sequences was isolated. pAlV-6C6 NE0 and other deletion mutants were partially digested with Sphl, mixed with the purified Sphl CAT fragment, ligated, and transformed E. co/i HE1 01. Transformants were selected on LB + chloramphenicol plates.

deletions at the 5’ end are given in Fig. 3. We have observed no difference between pATV-C6 and some other clones when the deletions extend up to -210, and therefore these are not shown. It is apparent that pATV-C6-26 whose 5’ boundary is -208 did not lose enhancer activity. However, when the deletion further extends to -201 (pATV-C6-8) 60-7096 of the activity is lost. Removal of an additional 21 nucleotides (C6-8) resulted in almost 85 to 90% loss of activity. These results suggest that the sequence between nucleotides -208 and -201 is required for maximal transcription. Cullen et a/. (3) also observed decreased activity when the sequence -272 to -138 was removed. However, in their studies, the smallest 5’ deletion is extended from -272 to -138 and therefore precise localization of the enhancer sequence was not possible. To further demonstrate the presence of a second domain between -208 and -201, a more sensitive vector system has been developed. Several groups showed that chloramphenicol transacetylase provide a convenient and more sensitive assay system to monitor transcription. In addition, this assay eliminates the possibility of contamination by plasmid DNA, which is a potential problem in RNA hybridizations. The results presented in Fig. 3 provide strong evidence for the requirement of the sequence between -208 and -201 for efficient transcription. We note that there is some discrepancy between the NE0 hybridization results and the CAT assays. For example,

245

we have observed only threefold reduction in the amount of neo-specific RNA in C6-1 compared to C6 or C6-26 but at least 20- to 30-fold reduction in CAT activity. This may be due to the sensitivity of these techniques and to some extent due to contamination of RNA preparations by plasmid DNA because digestion of the RNA samples with ribonuclease-free deoxyribonuclease considerably decreased the extent of hybridization. Since these inherent problems are eliminated in CAT assays, and since we obtained similar results by two different methods, the evidence strongly suggests the presence of a second motif at -208 to -201. In parallel studies, deletions at the Sphl site (-141) also indicated that the sequence around -141 to -1 19 is required for enhancer activity (Fig. 3). These results suggest that the sequences between -208 and -201 and between -141 and -119 constitute the enhancer, as observed in other enhancers (1). Whether they constitute a single enhancer unit or two different motifs remains to be determined. Previous results from several laboratories indicated enhancer activity in the sequence between -272 and -138 (3, 10, 12). The enhancer sequence we identified appears to be the same as domain B of Laimis et al. (10). Deletion of only a few nucleotides around the Sphl site (-141) severely curtailed transcription. These results are in agreement with those of others (3) and indicate the importance of the sequence around -140, which has a potential Z-DNA-forming structure (17). We have also observed that in many nonpermissive cells that the ASV LTR is modified by methylation around the sequence -1 13 and this modification reduced the levels of transcription of viral RNA (9). Recent evidence indicates that in vitro methylation of the sequence between -299 and -55 also greatly decreases the extent of transcription (Guntaka, Gowda, Wagner, and Simon, in press). Overall these results clearly identify a major enhancer domain in the LTR of the Prague C strain of Rous sarcoma virus. It is interesting to note that deletion of the sequence between either -208 and -201 or -141 and -119 resulted in loss of transcription. Recently it has been shown that two enhancer factors bind to two distinct domains in the LTR of SR-A RSV. These sequences, -229 to -203/-l 92 and -146 to -12 1, which were mapped by DNasel footprinting (24) correspond to the functional domains we have identified in this work. The sequence TTGGTGGTAG (-132 to -123), shares homology with the consensus core sequence XTGTGGAAAG, and is part of the first domain at -141 to -1 19. Some common cellular trans-acting factors might recognize this domain, whereas the second domain at -208 to -201, GTAGTCTTGC, which is unique to RSV LTR, may be specifically recognized by

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% Activity

D3A-Sph2

DJA-Sph

10

DJA-Sphl

NE0 -

CAT

100

100

100

68

35

4

15

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FIG.~. Effect of deletions on the transcription. The extent of deletions, as determined in Figs. 1 and 2, are drawn to the scale. Supercoiled DNAs were prepared by the CsCI-ethidium bromide gradients, and introduced into QT6 cells by the calcium phosphate method of Graham and van der Eb (5) as described previously (75). Approximately 10 pg DNA was used per loo-mm plate. After 5 hr, the cells were treated with 10% glycerol for 3 to 5 min and the cells were harvested after 28 to 40 hr. RNA was isolated by the CsCl method (2, 6). Serial dilutions ranging from 0.1 to 10 pg of total RNA were spotted onto nylon membranes, and the filters were baked and hybridized with the neo-specific probe. A 0.7-kb Pvull fragment specific for the neomycin gene was cloned in Ml 3mp7 and used as a probe after nick-translation (20) using [3zP]dCTP. The specific activities ranged from 50 to 100 X 10’ cpm/pg. The filters were hybridized in 50% HCONHp at 42” in a buffer containing 4x Denhardt’s buffer, 100 pg/ml yeast RNA, 100 pg/ml calf thymus DNA, and 10% dextran sulfate. The filters were washed at room temperature and exposed against X-ray filter at -70” for 2 to 4 days. Each spot was cut out and counted in a scintillation counter. RNA from normal QT6 cells was used as control and these spots were used to substract background from the experimental values in calculating the percentage activity. At the highest concentration, about 270 to 500 cpm were hybridized. Chloramphenicol transcetylase assays were done essentially as described by Gorman et al. (25) using [‘4C]chloramphenicol (New England Nuclear). C6-26 NE0 DNA transfected cell extracts were used as negative control. CAT assays were performed at two different protein concentrations which were determined by the Bio-Rad reagent using bovine serum albumin as standard, Conversion of [‘4C]chloramphenicol into its acetylated form was monitored by thin-layer chromatography using silica gel. Following chromatography, the spots were cut out and counted in a scintillation counter. At lower protein concentrations at least 60 to 70% of [“‘C]chloramphenicol in controls (C6) was converted to the acetylated forms. This is taken as 100% in computing the level of activity for various clones, The CAT assays for the Sphl deletions have not been done.

virus-coded factors or factors present only in permissive cells. Experiments to test these possibilities are in progress.

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ACKNOWLEDGMENTS We thank Drs. David Pintel and Mark McIntosh for comments on the manuscript and Peter Cleavinger for technical assistance with CAT assays. This work was supported by Grant CA36790 from the National Cancer Institute.

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