Characterization of the Jembrana disease virus tat gene and the cis- and trans-regulatory elements in its long terminal repeats

Abstract

Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDV tat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides -68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide -196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.

FIG. 1

Schematic representation of the JDV LTR promoter construct and the various derived deletion clones used for transient transfection analysis. The locations of the various promoter elements, U3, R, U5, the TATA box, and the putative TAR region, are indicated. Several predicted regulatory factor binding sites, for NF-κB, SP-1, AP-4, and the core enhancer element, are also shown. Solid lines represent the sequences retained in the LTR deletion plasmids. The 5′ and 3′ ends for each deletion plasmid are numbered with respect to the transcription start site (+1).

FIG. 2

Cotransfection of the JDV LTR with various amounts of JDV tat (pRSV-JTAT). pJDV-U3R was transfected into either FBL (0.5 μg of DNA) or CV-1 (1 μg of DNA) cells with varying amounts of pRSV-JTAT (0, 0.25, 0.5, 1.0, and 2.5 μg of DNA) by using Lipofectamine. The total amount of DNA used was kept constant by adjusting with pUC18 DNA. The fold activation, calculated as the average concentration of CAT protein (picograms of CAT protein per milligram of total cellular protein) in the presence of tat divided by the average concentration of CAT protein in the absence of tat, is shown above each bar.

FIG. 3

Northern blot analysis for CAT-specific transcripts. The analysis was carried out with a ³²P-labeled CAT-specific probe. The two CAT-specific RNA species indicated by arrows are the spliced and unspliced forms of the CAT mRNA (2). A GAPDH probe was used as an internal control to normalize the amount of RNA loaded onto each lane.

FIG. 4

(A) The predicted secondary structure of the putative JDV TAR region. RNA folding was performed by the RNAFOLD program in the GCG Wisconsin package. The entire R region of the JDV LTR was included in the analysis. The free energy for the folded structure is −44.8 kJ/mol. (B) Effects of site-directed mutagenesis within the first stem-loop structure of JDV TAR on tat transactivation. Based on the deletion clone p3D+53, we constructed a set of mutation constructs, each of which bears three nucleotide substitutions. The mutated nucleotides are boldfaced, and their corresponding positions in the stem-loop structure are shown. In each experiment, 0.5 μg of the wild-type plasmid (p3D+53) or 0.5 μg of each mutant plasmid was cotransfected into CV-1 cells with the JDV tat plasmid. At 48 h posttransfection, cells were lysed and the lysates were subjected to a CAT ELISA as described in Materials and Methods. The average fold activation, calculated as described in the legend to Fig. 2 and derived from three independent experiments, is presented for each plasmid.

FIG. 4

(A) The predicted secondary structure of the putative JDV TAR region. RNA folding was performed by the RNAFOLD program in the GCG Wisconsin package. The entire R region of the JDV LTR was included in the analysis. The free energy for the folded structure is −44.8 kJ/mol. (B) Effects of site-directed mutagenesis within the first stem-loop structure of JDV TAR on tat transactivation. Based on the deletion clone p3D+53, we constructed a set of mutation constructs, each of which bears three nucleotide substitutions. The mutated nucleotides are boldfaced, and their corresponding positions in the stem-loop structure are shown. In each experiment, 0.5 μg of the wild-type plasmid (p3D+53) or 0.5 μg of each mutant plasmid was cotransfected into CV-1 cells with the JDV tat plasmid. At 48 h posttransfection, cells were lysed and the lysates were subjected to a CAT ELISA as described in Materials and Methods. The average fold activation, calculated as described in the legend to Fig. 2 and derived from three independent experiments, is presented for each plasmid.

FIG. 5

(A) Transactivation of the JDV LTR by BIV Tat in CV-1 and FBL cells. The pJDV-U3R plasmid (1 μg of pJDV-U3R for CV-1 cells and 0.5 μg for FBL cells) and varying amounts of the BIV tat plasmid (0, 0.25, 0.5, 1.0, and 2.5 μg) were cotransfected into cells by using Lipofectamine as described in Materials and Methods. (B) Effects of HIV Tat on JDV LTR expression in CV-1 and FBL cells. The pJDV-U3R plasmid (1 μg for CV-1 cells and 0.5 μg for FBL cells) and varying amounts of the HIV tat plasmid (0, 0.25, 0.5, 1.0, and 2.5 μg) were transfected by using Lipofectamine, and the CAT protein concentration were determined. The fold activation is shown above each bar.

FIG. 6

Comparison of the effects of JDV Tat and BIV Tat on BIV LTR expression in either CV-1 (A) or FBL (B) cells. Cells were transfected with 0.5 μg of pBIV-LTR-CAT and varying amounts of the JDV tat plasmid (0, 0.25, 0.5, 1.0, and 2.5 μg) by using Lipofectamine. The fold transactivation is shown above each bar.

FIG. 7

Comparison of the effects of JDV Tat and HIV Tat on HIV LTR expression. CV-1 cells were transfected with 0.1 μg of pHIV-LTR-CAT and varying amounts of the JDV or HIV tat plasmid (0, 0.25, 0.5, 1.0, and 2.5 μg) by using Lipofectamine. The fold transactivation is shown above each bar.

FIG. 8

Alignment of amino acid sequences of JDV, BIV, HIV-2, and HIV-1 Tat exon 1. Peptide sequences were aligned with the GCG Pileup program. The putative N-terminal (N-TERM), cysteine-rich (CYS-RICH), core region, basic, and C-terminal (C-TERM) domains of Tat are underlined and labeled (24). Asterisks mark amino acids that are conserved in JDV and other Tat sequences.