Demonstration that orf2 encodes the feline immunodeficiency virus transactivating (Tat) protein and characterization of a unique gene product with partial rev activity

Abstract

The long PCR technique was used to amplify the three size classes of viral mRNAs produced in cells infected by feline immunodeficiency virus (FIV). We identified in the env region a new splice acceptor site that generated two transcripts, each coding for an 11-kDa protein, p11(rev), whose function is unknown. The small-size class of mRNAs included two bicistronic orf2/rev mRNAs and two rev-like mRNAs, consisting only of the second exon of rev and coding for a 15-kDa protein, p15(rev). p15(rev) contained the minimal effector domain of Rev and was sufficient to mediate partial Rev activity. The bicistronic mRNAs encoded two distinct proteins, one of 23 kDa corresponding to Rev and a 9-kDa protein encoded by the orf2 gene. The orf2 gene product is a protein of 79 amino acids with characteristics similar to those of the Tat (transactivator) proteins of the ungulate lentiviruses. Transient expression assays, using the FIV long terminal repeat (LTR) to drive transcription of the bacterial gene for chloramphenicol acetyltransferase demonstrated that the orf2 gene transactivates gene expression an average of 14- to 20-fold above the basal level. Deletion mutants of the FIV LTR were generated to locate sequences responsive to transactivation mediated by the orf2 gene. A 5' deletion mutant that removed the AP1 site resulted in residual low-level transactivation by orf2. Further experiments using LTR mutants with internal deletions identified three regions located between positions -126 and -47 relative to the cap site that were important for orf2-directed transactivation. These regions include the AP1 site, a C/EBP tandem repeat, and an ATF site.

FIG. 1

Transcriptional map of FIV. A schematic representation of the FIV genome is shown at the top. The exon structure and locations of splice donor (SD) and splice acceptor (SA) sites were determined by sequencing the PCR-amplified cDNAs. The arrows represent locations and orientations of the oligonucleotide primers used in this study. The viral transcripts are deduced from the sequence of the cDNA clones. Each transcript is designated by its exon composition. Transcripts 1.3.5 and 1.2.3.5 are newly described in this study.

FIG. 2

Long PCR amplification of the different size classes of FIV transcripts. PCR products were resolved by electrophoresis on 1.3% agarose gel and stained with ethidium bromide. The sizes of the HindIII-cut λ DNA markers (outside lanes) are indicated in kilobases. (A) Optimization using different enzyme ratios. Taq and Deep-Vent polymerases were used alone (lanes 1 and 2) or in combination (lanes 3 to 12), where 5 U of Taq was mixed with serial twofold dilutions of Deep-Vent, starting with 0.5 U of Deep-Vent. (B) PCR amplification with the primer pairs LA4-LA7 (left panel) and LA4-LA11 (right panel). Numbers on the left indicate the exon composition of each PCR product, deduced from the sequence of the cDNA clones.

FIG. 3

Coding potential of the orf2/rev and rev-like cDNAs. The cDNA clones of the orf2/rev and rev-like transcripts were analyzed by an in vitro coupled transcription-translation system (Promega). ³⁵S-labeled proteins were immunoprecipitated by an anti-orf2 rabbit serum (A), an anti-Rev rabbit serum (B to D), and an FIV-positive cat serum (C). Immune complexes were resolved by electrophoresis on a sodium dodecyl sulfate–10 to 20% polyacrylamide gel. Sizes of the protein molecular weight markers are indicated in kilodaltons on the left. The cDNA clones used as templates in the translation reactions are indicated above the lanes.

FIG. 4

Rev activity of the orf2/rev and rev-like transcripts. (A) Schematic structure of Rev, p11^rev, and p15^rev proteins. (B) Each rev and rev-like cDNA clone was cotransfected with a CAT-RRE vector in CrFK cells. Transfected cell lysates were assayed for CAT activity as described in Materials and Methods. Relative CAT activity is the mean value of two independent experiments performed in triplicate; standard deviations are shown as error bars. The relative increase of CAT activity compared to the mock control for each construct is shown on the right.

FIG. 5

The orf2 gene encodes a transactivator of the FIV LTR. (A) Schematic organizations of the visna virus Tat protein and of the FIV-PPR and FIV-34TF10 orf2 gene products. (B) Each cDNA clone indicated on the left was cotransfected with an FIV LTR-CAT vector in CrFK cells. (C) Comparison of transactivation of the FIV LTR mediated by the PPR and 34TF10 orf2 genes in CrFK and HeLa cells. The amount of pFIV LTR-CAT vector is indicated in parentheses. The CAT activity of the transfected cells was assayed as described in Materials and Methods. The experiment were repeated at least twice, in triplicate; standard deviations are shown as error bars. The relative increase of CAT activity compared to the mock control for each construct is shown on the right.

FIG. 6

Deletion of the LTR sequences required for transactivation. (A) Deletion mutants of the FIV LTR promoter. The putative cis-acting regulatory elements contained in the U3 region of the LTR are indicated at the top. The panel of 5′ deletion mutants was previously described (35) and kindly provided by J. F. Thompson and colleagues. wt, wild type. (B and C) Transactivation of 5′ deletion mutants (B) and internal deletion mutants (C) of the FIV LTR by PPR orf2. Each deletion mutant was cotransfected in CrFK cells with the PPR orf2 vector (gray bars). Cotransfection of each mutant with pCR3 defined baseline activity (blank bars). CAT activity was assayed as described in Materials and Methods. The experiment was repeated twice, in triplicate; standard deviations are shown as error bars. The percentage of wild-type LTR activity of each deletion mutant in response to transactivation by orf2, as well as the relative increase of CAT activity compared to the basal level for each deletion mutant, is shown on the right.