Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs

Abstract

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

FIG. 1

Organization and splicing patterns of EIAV. Schematic of EIAV genome with open reading frames (ORFs). The tat ORFs are indicated with a t, and the first exon of rev is marked with an r (location in genome indicated by the shaded region). Splicing patterns and genes expressed are indicated. The ttm ORF encodes a truncated transmembrane protein of unknown function (3). LTR, long terminal repeat.

FIG. 2

Exon 3 splicing requires the purine-rich sequence. (A) Diagram of RNA substrates used for in vitro splicing, showing the locations of exons 2 and 3. All substrates contain the exon 2 splice donor and exon 3 splice acceptor. The approximate location of the purine-rich sequence is highlighted. (B) After incubation for 2 h in HeLa cell splicing extracts, RNA products were electrophoresed through 4% polyacrylamide gels and visualized by autoradiography. The locations of spliced and unspliced products are shown. The fastest-migrating products in lanes 3 to 5 are intron products resulting from splicing. Sizes are shown at the left (in nucleotides).

FIG. 3

Rev inhibits exon 3 splicing. GST-Rev or GST was added at the indicated concentrations to the splicing reaction mixtures. The locations of the splicing products are indicated on the right. Sizes are shown at the left (in nucleotides).

FIG. 4

GST-Rev binds the ESE in exon 3. (A) Locations of the RNA probes, relative to exon 3, used in RNA gel mobility shift assays. The purine-rich ESE sequence is highlighted. SA, splice acceptor; SD, splice donor. (B) After incubation with GST or GST-Rev, radiolabeled RNAs were electrophoresed through 8% native polyacrylamide gels. The locations of GST-Rev-RNA complexes are indicated with arrows. (C) Competition assays were performed with either 0.5 or 1 μg of the indicated excess of unlabeled competitor RNAs. Competitors were mixed with GST-Rev 10 min prior to the addition of radiolabeled probe.

FIG. 5

In vitro splicing and RNA binding of ESE mutants. (A) Sequence of two purine stretches (designated A and B) in exon 3. GAA repeats were mutated to GCA in the largest splicing construct (Fig. 2A) and RNA probe RREp4 (Fig. 4A). (B) In vitro splicing analysis of mutant ESE constructs. The locations of splicing products are indicated. Sizes are shown at the left (in nucleotides). (C) RNA gel mobility shift assays detecting GST-Rev binding to the mutant probes. The arrow points to the location of shifted RNAs.

FIG. 6

EIAV ESE can function as an RRE. (A) pDM138-derived reporter vectors containing various regions of the EIAV genome were used in transient transfections with and without Rev, and CAT assays were performed in 293 cells as described in Materials and Methods. The results are presented as the percent acetylation. Experiments were performed in triplicate, and the results represent the mean of at least nine independent transfections. Error bars denote the standard error of the mean. (B) The ESE mutations indicated in Fig. 5A were also introduced into the ERRE-1A reporter vector and assayed for CAT activity in the presence and absence of Rev as described for panel A.

FIG. 7

SF2/ASF inhibits Rev-dependent nuclear export. pERRE-1 reporter plasmid was cotransfected with 0.5 ng of pRevWT and increasing amounts of pSF2/ASF. CAT levels were quantified by ELISA and are reported as picograms of CAT per normalized lysate. Results represent the mean of six independent transfections, and the error bars denote the standard error of the mean. Asterisks indicate values significantly different (P < 0.05) from control transfections which contained no pSF2/ASF.