Characterization of functional domains of equine infectious anemia virus Rev suggests a bipartite RNA-binding domain

Abstract

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.

FIG. 1.

Organization and splicing patterns of EIAV and the EIAV Rev amino acid sequence. (A) Schematic of the EIAV genome showing open reading frames (ORF) and predominant mRNAs (a to e) isolated from virus-infected tissue culture cells (27). Regulatory proteins Tat (T) and Rev (r, rev) are translated from the four-exon mRNA (a). In the presence of Rev, EIAV exon 3 is skipped, resulting in a three-exon multiply spliced mRNA that encodes only Tat (b). Structural proteins and progeny RNA molecules are translated from singly spliced (d) and unspliced mRNAs (e). Ttm, a protein of unknown function (5), is encoded by a two-exon mRNA (c). (B) The amino acid sequence of the wild-type EIAV Rev H21 (7) is shown and numbered 1 to 165. The nuclear export signal (aa 31 to 55) is boxed.

FIG. 2.

Functional analyses of Rev deletion mutants. (A) Terminal and internal deletions in Rev cDNA were generated from pRevWT by PCR-ligation-PCR as described in the text. The region of amino acids deleted in each cDNA is indicated. (B) Activity of Rev deletion mutants was assayed in transient expression assays. Results are reported as activity relative to pRevWT. Cells transfected with the pCR3.1 vector DNA were used as the negative control (Neg.) in all assays. Error bars denote the standard error of the mean. Graph 1 shows nuclear export activity measured using a CAT reporter assay (7). Lysates were normalized by β-galactosidase activity, and CAT activity was measured as the percentage of acetylation. Individual experiments included triplicate wells, and the data shown represent the means of at least three separate experiments. Graph 2 shows supernatant reverse transcriptase (RT) activity following trans-complementation of a Rev-defective clonal cell line, Cf2Th/51, with Rev deletion mutants. Wt, wild type.

FIG. 3.

The KRRRK motif in the C terminus of Rev is required for nuclear localization. (A) Amino acid sequence of the C terminus of wild-type Rev (aa 144 to 165) and the location of deletions or alanine substitutions introduced into wild-type GFP-Rev mutants. (B) Subcellular location of GFP-Rev deletion mutants (GFP-RDM13 and GFP-RDM14) and GFP-Rev containing alanine substitutions in the C terminus. Cf2Th cells were transfected with the specified Rev-GFP cDNAs in the presence or absence of 5 nM leptomycin B (LMB). Images of fixed and stained Cf2Th cells were obtained by confocal laser microscopy. Brightness and contrast were adjusted with Adobe Photoshop 4.0.

FIG. 4.

Identification of sequences critical for RNA-binding activity of EIAV Rev. (A) MBP-Rev deletion mutants (MRD) containing 5′ or 3′ deletions used to map regions of Rev required for RNA binding. The Rev amino acids retained in each construct are indicated. (B) Expression and purity of MRD mutants were assessed by Coomassie staining of SDS-PAGE gels. The molecular size of each deletion mutant was confirmed using molecular size (MW) markers. (C) RNA-binding activity of MBP-Rev fusion proteins was determined by UV cross-linking and SDS-PAGE. Rev fusion proteins were incubated with radiolabeled EIAV RRE (nucleotides 5443 to 5565), cross-linked with UV irradiation, and treated with RNase. RNA-protein complexes were separated by SDS-PAGE and quantified using a PersonalFX scanner and Quantity One software (Bio-Rad). Negative controls included bovine serum albumin (BSA), MBP, and antisense RRE. (D and E) RRE-binding activity of Rev deletion mutants as described for panel C.

FIG. 5.

RRE-binding activity and functional analyses of Rev mutants. A. Identification of specific motifs essential for RRE-binding activity of EIAV Rev. Point mutations introduced into the RRDRW, ERLE, and KRRRK motifs include alanine substitution of charged and/or Arg residues (AADAA, AALA, and KAAAK) and substitution of Asp for Leu in the ERLE motif (L95D). Binding activity was assessed as described in the legend to Fig. 4C. B. Activity of Rev point mutation mutants (AADAA, AALA, KAAAK, and L95D) and deletion mutant (RDM13) were assayed as described in the legend to Fig. 2B. Neg., negative control. Wt, wild type.