Localization of Sequences Responsible for trans ... - Journal of Virology

4 downloads 0 Views 2MB Size Report
Chakrabarti, L., M. Guyader, M. Alizon, M. D. Daniel, R. C. Desrosiers, P. Tiollais .... Rosen, C. A., J. G. Sodroski, L. Willems, R. Kettmann, K. Campbell, R. Zaya ...
JOURNAL

OF

Vol. 62, No. 1

VIROLOGY, Jan. 1988, p. 120-126

0022-538X/88/010120-07$02.00/0

Localization of Sequences Responsible for trans-Activation of the Equine Infectious Anemia Virus Long Terminal Repeat LEVANA SHERMAN,' ARNONA GAZIT,' ABRAHAM YANIV,' TOSHIAKI KAWAKAMI,2 JOHN E. DAHLBERG,2 AND STEVEN R. TRONICK2* Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel 69978,1 and Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 208922 Received 1 June 1987/Accepted 24 September 1987

We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome. The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters. In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced. Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced. trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR. EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775). Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product.

Based on a number of criteria, including pathogenesis, replication in vitro, morphology, antigenic reactivity, and predicted amino acid sequence similarities, the equine infectious anemia virus (EIAV) and the human immunodeficiency virus (HIV) are considered members of the lentivirus subfamily of retroviruses (reviewed in reference 25). The hallmarks of lentivirus infections are virus persistence in the face of a host immune response and induction of chronic disease. As part of our efforts to understand the molecular basis of lentivirus pathogenicity, we have recently determined the complete nucleotide sequence of an integrated EIAV genome (28). Our studies and those of others (12, 43, 51) have revealed additional similarities between EIAV, prototype lentiviruses, and HIV, but also differences. For example, the size (321 bases) of the EIAV long terminal repeat (LTR) is much closer to those of visna virus and caprine arthritis encephalitis virus (CAEV) (26, 27, 43, 45) LTRs than to that of HIV (645 bases). For all of these viruses, the gag and pol genes overlap but pol and env do not. The EIAV env open reading frame encodes a precursor polypeptide that is similar in size to those of visna virus and HIV isolates and shows significant amino acid sequence similarity to their predicted transmembrane proteins (28). The EIAV env open reading frame terminates before the LTR, as is the case for HIV, whereas in the visna virus genome these sequences overlap it. Perhaps the most striking difference observed is the presence in visna virus and HIV of an open reading frame (designated Q and sor, respectively) between the pol and env genes (reviewed in reference 25) for which an equivalent has not been observed in the EIAV clones sequenced to date (28, 43). A short 3' open reading frame (ORF) is present in visna virus and HIV (3'-ORF) that overlaps the -LTR, whereas EIAV harbors a 3' ORF of similar coding capacity, but it terminates upstream of the 3' LTR. A striking property of visna virus and HIV is their ability to activate expression of their genomes via a virus-coded

trans-activating protein (1, 26, 50). For HIV, evidence has been obtained that indicates that trans-activation occurs via interaction of this protein, designated tat (for trans-acting transcriptional regulation) with certain LTR sequences (TAR, trans-activating responsive sequence) (31, 39). This interaction has been shown to be required for virus replication (9, 15). Computer comparisons of the HIV tat ORF indicated that such a sequence might be present in EIAV in a region analogous to that in which the first coding exon of the HIV tat gene resides (28). To determine whether transactivation may be involved in the replication cycle of EIAV, we used the chloramphenicol acetyltransferase (CAT) transient expression assay (21, 22) to assess the transcriptional activity of the EIAV LTR and its ability to respond to viral gene products. Cells that were infected with EIAV or transfected with a molecularly cloned EIAV genome were found to contain increased amounts of both cat-specific mRNA and enzymatic activity compared with control cells. By analyzing deletion mutants, the viral sequences responsible for trans-activating effects were localized to a region between pol and env. We were also able to obtain evidence suggesting that this trans-activating factor interacts with LTR sequences corresponding to the 5' terminus of the viral RNA. MATERIALS AND METHODS Plasmid constructions. The construction of the various EIAV-CAT and CAEV-CAT plasmids is described in the legend to Fig. 1. The recombinant cat plasmids pSV2-CAT, pSVO-CAT, and pRSV-CAT have been described elsewhere (21, 22). pEIAV (p1369), which contains the EIAV provirus DNA, was described previously (55). The deletion mutants obtained from this plasmid are described in the legend to Fig. 1. The final plasmid constructions were confirmed by extensive restriction enzyme mapping. Cell cultures and transfections. Canine fetal thymus cells (Cf2Th) (8) were grown at 37°C in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. Transfection and CAT assays. Cells were plated into 100-

* Corresponding author. 120

trans-ACTIVATION OF EIAV LTR

VOL. 62, 1988 Bgl 11

L~~~~~~~~~~~~

pEIAV LTR-CAT

I

Nco I

Barn

H

Bgl 11 pElAV U3A R-CAT r

U3

Nco I

A

i

Hinf

Bgl 11 *-

pCAEV LTR-CAT

--I Pat

II

U3

I

R

I U5

Al

hyde-agarose gels and transfer of RNA to nitrocellulose filters which were hybridized with a 32P-labeled cat gene DNA probe (22). Hybridizations were carried out at 42°C for 48 h in a solution containing 50% formamide, 5 x SSC (1 x SSC is 150 mM NaCI plus 15 mM sodium citrate), 4 x Denhardt solution, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (SDS), and 200 ,ug of denatured salmon sperm DNA per ml. Following hybridization, filters were washed at room temperature for 1 h with 2 x SSC-0.1% SDS and then at 50°C for 1 h with 0.1 x SSC-0.1% SDS and then exposed to X-ray film.

Pvu 11

FIG. 1. EIAV and CAEV LTR-CAT constructs. DNA fragments containing LTR sequences were isolated by digesting cloned viral DNAs with the indicated restriction enzymes and filling in staggered ends with either E. coli DNA polymerase I (Klenow fragment) or T4 DNA polymerase and were then inserted into the BglII site (also filled in) of pSVO-CAT (21). The BamHI site in EIAV is located (position 387 [28]) 66 bases downstream from the 3' border of the 5' LTR. The Hinfl site in the EIAV LTR is located 3 nucleotides downstream from the putative cap site (position 230 [28]). The PvuII site used for the construction of the CAEV-CAT plasmid resides 2 bases downstream from the LTR border. The wavy and solid lines represent flanking upstream cellular and downstream viral sequences, respectively.

(-1.5 x 106 cells) or 60-mm (8 x 105 cells) tissue culture dishes the day before transfection. Subconfluent (-80%) cell monolayers were transfected with plasmid DNA by a modification of the calcium phosphate coprecipitation technique (21-23). Cultures were treated with the DNA precipitate for 24 h. Cells were then treated with 20% glycerol in serum-free DMEM for 2 min. The glycerolcontaining medium was replaced with fresh DMEM, and the cells were incubated for another 24 h. All transfected cultures were maintained at 37°C with 5% CO2. Cells were harvested 48 h after transfection, and extracts were prepared by three 2-min freeze (liquid nitrogen) and thaw (37°C) cycles and then briefly centrifuged to remove cell debris. The CAT assay was performed by a published procedure (21). Briefly, extracts were incubated for 10 min at 60°C before the assay. Reactions were performed using 5 to 120 ,ul of extract (-20 to 625 ,ug of protein) in a 145-pul reaction mixture containing 0.2 ,uCi of [14C]chloramphenicol and 4 mM acetyl-coenzyme A in 0.25 M Tris hydrochloride (pH 7.8) at 37°C for 1 to 2 h. The [14C]chloramphenicol was separated from the acetylated forms by ascending thin-layer chromatography. Radioactive reaction products were located by autoradiography and cut out from the chromatograms to determine levels of acetylated and unacetylated compounds. Under the conditions described, the assay was linear with respect to time and protein concentration, until about 70% conversion was achieved. The levels of CAT activity increased as a linear function of the amount of construct DNA used to transfect cells up to 6 ,g (-3 pmol) of DNA per 8 x 105 cells. When the amount of construct DNA was varied, the total amount of DNA used for transfection was adjusted to 10 ptg with pBR322 DNA. At least two replicate determinations were made, and the results were found to differ by not more than 10%. Analysis of RNA. Total RNA was extracted from the transfected cells by a guanidine hydrochloride lysis procedure followed by pelleting through a cushion of 5.7 M CsCl (6). For quantitation of RNA, dot blots were done as described previously (44). Northern (RNA blot) analysis of cat-specific mRNA was performed by using 1.5% formaldemm

121

RESULTS Promoter activity of EIAV LTR. Sequences conforming to those of transcriptional control elements are present within the U3 region of the EIAV LTR (12, 28, 43). To establish the effects of the EIAV LTR on gene transcription, we examined its ability to drive the expression of the bacterial cat gene. Recombinant cat plasmids were constructed (Fig. 1) in which either the entire EIAV LTR (pEIAV LTR-CAT) or the putative promoter region, which includes U3 and three nucleotides of the R region (designated pEIAV U3AR-CAT), were placed 5' to the cat gene. For comparison, other promoters were also tested. Thus, we constructed plasmids in which the cat gene was linked to the CAEV LTR (45) (pCAEV LTR-CAT), to the Rous sarcoma virus (RSV) LTR (21) (pRSV-CAT), and to the simian virus 40 (SV40) earlyregion promoter (pSV2-CAT) (22). The plasmid pSVO-CAT containing no promoter (22) was used as a control. cat plasmid DNAs were introduced into a canine cell line (Cf2Th) in which EIAV replicates readily. Both EIAV-CAT plasmids (pEIAV LTR-CAT and pEIAV U3AR-CAT) directed detectable, though low, levels of CAT activity which reached a plateau at about 6 ,ug of construct DNA per 8 x 105 cells (3.3 pmol) (Table 1). In contrast, CAEV and RSV LTRs and the SV40 promoter were much more efficient in directing CAT activity (25- to 90-fold) (Table 1). EIAV LTR is trans-activated in cells containing the EIAV genome. To examine whether the EIAV genome codes for trans-acting factors that influence EIAV LTR activity, the transient expression of the cat gene under the control of the EIAV LTR was measured in EIAV-infected canine cells. Data from a representative experiment (Table 1) reveal a significant elevation (>50-fold) of EIAV LTR-directed gene expression in EIAV-infected cells compared with uninfected cells. In other experiments (not shown), about 50- to 100-fold increases in activity were observed. In contrast, the activity of pEIAV U3AR-CAT was not stimulated in EIAV-infected cells. To provide additional evidence for the viral specificity of trans-activation, a molecularly cloned EIAV genome was used. CAT activity was elevated >100-fold when dog cells were cotransfected with 1 jig each of pEIAV-LTR and pEIAV DNA (Table 1). Evidence that the effect was specifically due to the presence of the EIAV genome was provided by the findings that CAT expression was augmented as a function of increasing EIAV DNA used for transfection (data not shown). cat expression driven by the CAEV LTR, RSV LTR, or SV40 promoters was not stimulated in canine cells cotransfected with EIAV DNA. This suggested that the trans-activating effects observed were specific for the EIAV LTR. Alternatively, these promoters might have been maximally stimulated in these cells and addition of an EIAVencoded factor(s) could have had no enhancing effect. We consider it unlikely, however, that three different promoters would behave in this manner.

122

SHERMAN ET AL.

J. VIROL.

trans-Activation of the EIAV LTR results in increases in both transcription and translation. In an effort to determin( the mechanism(s) of LTR activation by the EIAV genome, steady-state levels of cat transcripts, as well as CAT enzyme activity, were determined in cells either infected with EIAV or cotransfected with cloned EIAV DNA. As demonstrated by Northern blot analysis (Fig. 2A) and dot blot quantitation (Fig. 2B), the levels of cat mRNA directed by the EIAV LTR were higher in EIAV-infected and EIAV cotransfected cells than in uninfected cells. Hybridization of identical RNA dot blots to an actin probe confirmed the application of equal amounts of RNA (Fig. 2B, lanes c and d). The stimulation of the levels of cat mRNA was specifically due to the EIAV LTR, since no differences were observed in cells transfected with the SV40 promoter construct (Fig. 2B, lanes e and f). Whereas the steady-state levels of cat mRNA were increased by about 8-fold in EIAV-infected cells (Fig. 2B, lanes a and b) and 16-fold in cells cotransfected with EIAV DNA (Fig. 2B, lanes g and h), CAT enzyme activity was increased by more than 80-fold (Fig. 2C). Thus, the steadystate level of EIAV mRNA, although significantly increased in cells containing the EIAV genome, was not sufficient to account for the overall enhancement of CAT activity. Localization of sequences encoding the trans-acting factor. To determine the region(s) of the EIAV genome essential for its trans-acting effects, deletions were introduced into various parts of cloned EIAV (p1369) DNA (55). These deletion mutants were then tested for their ability to stimulate cat expression in trans upon cotransfection with EIAV LTRCAT. To ensure accurate measurement of the effect, each deletion mutant was tested by using increasing amounts of DNA in the presence of two constant amounts of the indicator DNA. The various deletion mutants are depicted in TABLE 1. Promoter activity and trans-activation of the EIAV LTR

Construct

pEIAV LTR-CAT pEIAV U3AR-CAT pCAEV LTR-CAT pRSV-CAT pSV2-CAT

CAT activity in Cf2Th cellsa

7.0 (0.04) 7.2 (0.05) 570 (3.56) 280 (1.75) 160 (1.00)

CAT activity of cells containing EIAV genomes relative to that of untreated cells Cf2Th cells infected with EIAVb

cotransfected with EIAV DNAC

Cf2Th cells

57.3 1.2 1.2 1.0 0.0

110 1.2 1.1 0.9 1.1

a 10 ,ug of DNA of the recombinant CAT plasmids depicted in Fig. 1 and also pSV2-CAT and pRSV-CAT were transfected into the indicated cell culture (1.5 x 106 cells per 100-mm dish). CAT assays were performed on extracts prepared 48 h after transfection. Enzyme activities were measured after 2 h of incubation, with 20 to 600 jig of protein used to establish the linear range of the assay. Data (obtained from the linear range) are expressed as the ratio of the radioacivity of acetylated forms of chloramphenicol divided by that of the unreacted substrate multiplied by 100 per 200 ,ug of protein. The results represent the average of at least two independent transfection assays and have been corrected for the CAT activity of pSVO-CAT (o

^

B.

jg RNA 48

i.

.: :@ e>.-

:

-:) ' :,-:'

24

12

6

3

C # * * *

*

.:.

s ', 8 b

a

7