Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus

Abstract

Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

FIG. 1.

Schematic diagram showing the RNA determinants for EIAV Gag-Pol frameshifting. The 58 nucleotides correspond to the coding sequence of C-terminal NC spanning from nucleotide 1707 to 1764 in EIAV_uk proviral DNA (AFO16316 [reference 9]). The slippery sequence is boxed, nucleotides involved in the GC-paired stretch are bolded, and the starting point of the p9 protein is indicated. The stem-loop structure was predicted by the stability of the RNA structure (18), and the pseudoknot structure was predicted based on computer-aided comparisons of a number of viral mRNAs (31).

FIG. 2.

Replication profiles and Gag-Pol expression of chimeric EIAV containing variant slippery sequences. (A) EIAV_uk provirus containing the indicated mutations was transfected into ED cells. Supernatant medium of each transfected sample was collected at the indicated days posttransfection (dpt). RT activity of the collected supernatant was assayed as a measure of virus production from transfected cells. Slippery sequences and the origin of each mutant are indicated. Duplicates of each mutant were transfected, and the presented data are representative of three independent experiments. (B) Cellular lysates of Cos-1 cells transfected with CMV_uk pro⁻ proviruses carrying the indicated mutants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 3 to 8% gradient Tris-acetate gel (see Materials and Methods). EIAV-specific proteins were identified by immunoblotting with a reference immune serum from a naturally infected horse (8, 24). The data represent at least duplicate experiments. (C) Digitally quantified frameshifting efficiencies of the mutants from the Western blotting, with the mean value of duplicate wild-type controls set as 100%, using a Kodak imaging station (see Materials and Methods). The data presented here were calculated from at least duplicate samples.

FIG. 3.

Function of GC-paired stretch in EIAV translational frameshifting and viral replication. (A) Predicted secondary structures of mutants altering the GC-paired segment. The slippery sequences are boxed, and mutated nucleotides are in bold type and underlined. (B) Replication profiles of EIAV mutants in ED cells transfected with proviral constructs described above for panel A compared to those of wild-type virus and a replication-defective slippery sequence mutant (A→T in Fig. 1). (C) Gag and Gag-Pol protein expressed in Cos-1 cells transfected with CMV_uk pro⁻ proviruses containing the indicated mutants, using procedures described in the legend for Fig. 2. (D) Relative frameshifting efficiencies of the mutants compared with wild-type control quantified as outlined in the legend for Fig. 2. The data are representative of three independent experiments.

FIG. 4.

Role of the predicted stem-loop structure in EIAV Gag-Pol frameshifting. (A) Predicted secondary structures of EIAV mutants. The slippery sequences are boxed, and mutated nucleotides are in bold type and underlined. (B) Replication profiles of EIAV proviral mutants defined in panel A after transfection of ED cells using procedures described in the legend for Fig. 2. (C) Analyses of Gag and Gag-Pol polyprotein expression in Cos 1 cells transfected with the indicated mutant constructs and compared to wild-type or a replication-defective (A→T) slippery sequence mutant. (D) The relative frameshifting efficiency of each mutant was analyzed as described in the legend for Fig. 2D. The data are representative of three independent experiments.

FIG. 5.

Function of the pseudoknot structure in EIAV translational frameshifting. (A) Predicted pseudoknot structures of indicated EIAV mutants. The slippery sequences are boxed, and mutated nucleotides are in bold type and underlined. (B) EIAV Gag and Gag-Pol polyprotein expression in Cos-1 cells transfected with CMV_uk pro⁻ proviruses containing the indicated mutants, as described in legends for previous figures. (C) Relative translational frameshifting efficiencies of the mutants compared with the wild type, calculated as described in the legend for Fig. 2. The data are representative of duplicate experiments.