Feline immunodeficiency virus OrfA is distinct from other lentivirus transactivators

Abstract

The feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning.

FIG. 1.

OrfA sequence comparisons. (A) Published sequences of OrfA from different FIV isolates were compared by using the CLUSTALW multiple alignment algorithm. (B) Chou-Fasman and Robson-Garnier consensus secondary structure-forming potential for PPR-OrfA and TM-2 OrfA are compared to show distinctions.

FIG. 2.

Purification of OrfA. (A) Western blot (lane 1) and Coomassie blue-stained gel (lanes 2 and 3) of purified OrfA showing the presence of both the 9- and the 18-kDa forms (lane 1) and the membrane-purified 9-kDa (lane 2) and its dimeric form (lane 3) analyzed by SDS-10 to 20% PAGE. (B) Dissociation of the OrfA dimer in the presence of 0.5 M β-mercaptoethanol (lane 5) and with simultaneous boiling (lane 6) or neither of these treatments (lane 4), as detected by Western blot analysis with anti-OrfA antibody. Lanes 1 to 3 show identical treatments of the 9-kDa OrfA protein.

FIG. 3.

Gal4-OrfA assay. (A) PCR strategy followed for generating the Gal4-OrfA deletion clones by PCR-ligation-PCR (see Materials and Methods for details). (B) Diagram of the deletion clones of OrfA fused in frame with the Gal4 DNA-binding domain generated by the strategy describe in panel A. (C) Autoradiogram of the various deletion constructs expressed in vitro under the T7 promoter, immunoprecipitated with anti-OrfA polyclonal serum, and resolved by SDS-10 to 20% PAGE. (D) Transactivation of the G₅-E₁b-CAT target construct by the different Gal4-OrfA deletion constructs and appropriate controls.

FIG. 3.

Gal4-OrfA assay. (A) PCR strategy followed for generating the Gal4-OrfA deletion clones by PCR-ligation-PCR (see Materials and Methods for details). (B) Diagram of the deletion clones of OrfA fused in frame with the Gal4 DNA-binding domain generated by the strategy describe in panel A. (C) Autoradiogram of the various deletion constructs expressed in vitro under the T7 promoter, immunoprecipitated with anti-OrfA polyclonal serum, and resolved by SDS-10 to 20% PAGE. (D) Transactivation of the G₅-E₁b-CAT target construct by the different Gal4-OrfA deletion constructs and appropriate controls.

FIG. 4.

Analysis of domains within OrfA. (A) Diagram of the different OrfA deletion constructs used in the present study is shown. (B) Autoradiogram of the various deletion constructs expressed in vitro under the T7 promoter, immunoprecipitated with anti-OrfA polyclonal serum, and resolved by SDS-10 to 20% PAGE.

FIG. 5.

Effect of altering the consensus Kozak sequence in the bicistronic message on the relative expression levels OrfA and Rev. (A) The placement of the Kozak sequence (middle) and its modification (bottom) in the context of the original OrfA sequence (top) is depicted in cartoon form. (B) A total of 1.5 μg each of the Kozak-OrfA-Rev (lanes 1 and 2), Kozak (−1)-OrfA-Rev (lanes 3 and 4), and OrfA-Rev (lanes 5 and 6) constructs were expressed in vitro, and 50% of the lysate were immunoprecipitated with either anti-Rev polyclonal serum (lanes 1, 3, and 5) or anti-OrfA polyclonal serum (lanes 2, 4, and 6). The autoradiogram of the immunoprecipitated products analyzed by SDS-10 to 20% PAGE is presented. The fold levels of the expression with respect to the original sequence are presented on the right of each construct in panel A. (C) Putative hairpin structure at the end of the OrfA reading frame and preceding that of Rev.

FIG. 6.

Generation of OrfA-expressing cell lines. (A) Schematic of the FLAG-tagged OrfA construct coexpressing the neomycin phosphotransferase gene for resistance to G418. (B) The putative OrfA product from nuclear extracts of metabolically labeled C-5-9 cells (lane 2) immunoprecipitated with anti-OrfA antiserum was compared to identically treated CrFK cells (lane 1). (C) CAT assay of the same cell line (C-5-9) used in panel B but transfected with the FIV LTR-CAT construct and assayed for relative CAT levels. As a control, a construct (shown as -LTR) having the CAT gene but lacking the LTR was used to determine background CAT levels for each of these cell lines. In addition, simultaneous cotransfections of CrFK cells with the same FIV-LTR_WT-CAT and the FLAG-OrfA constructs were done to serve as a control. The CAT activity from C-4-3, a cell line expressing higher levels of OrfA but exhibiting a high rate of cell death, is also shown.

FIG. 7.

Modulation of LTR activity by OrfA-expressing cell lines. (A) CAT assay of CrFK cells or the C-5-9 cell line transfected with FIV-LTR_WT-CAT or FIV-47LTR-CAT constructs and assayed for relative CAT levels. Simultaneous cotransfection of CrFK cells with the same FIV-LTR_WT-CAT constructs with or without FLAG-OrfA were performed as a control. (B) CAT activity in the OrfA-expressing cell line C-5-9 transfected with FIV-LTR_WT-CAT (WT) or FIV-47LTR-CAT (−47) or having various site-specific deletions (δAP1, δC/EBP, δNF1, and δATF).

FIG. 8.

Stable expression of OrfA in CrFK and 104-C1 cells. (A) Western blot analysis of equal amounts of lysates from 104-C1 (lanes 1 to 3) or CrFK (lanes 4 to 6) either mock transduced (lanes 1 and 4) or transduced with GFP (lanes 2 and 5) and OrfA-GFP (lanes 3 and 6) with the anti-GFP monoclonal antibody (JL-8; Clontech). (B) Immunoprecipitation with anti-OrfA polyclonal serum (lanes 1 and 2) and anti-GFP antibody (lanes 3 and 4) from equal amounts of cell lysates of 104-C1 cells transduced with either GFP (lanes 1 and 3) or OrfA-GFP (lanes 2 and 4) metabolically labeled with ³⁵S-labeled methionine and cysteine. The arrows point to the positions of OrfA and GFP in the autoradiogram. (C) Micro-RT activity assay of CrFK cells transduced with either GFP or OrfA-GFP infected with 34TF10 or mock infected. The values represent RT activity within the first 5 days postinfection.