Biological characterization of Rev variation in equine infectious anemia virus

Abstract

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.

FIG. 1

Organization of the EIAV genome. (A) Known ORFs and predominant mRNAs isolated from virus-infected tissue culture cells (19). LTR, long terminal repeat. (B) Location of EIAV regions inserted into pDM138 CAT constructs (17). pERRE-All contains nucleotides 5280 to 7534, pERRE-1 contains a short 5′ RRE sequence overlapping the first Rev exon (nucleotides 5280 to 5834), and pERRE-2 contains a major portion of the remaining downstream EIAV sequence present in pERRE-All (nucleotides 5837 to 7534) (15). All numbering of nucleotides in the present report is based on that of Kawakami et al. (19).

FIG. 2

Amino acid sequence alignments of the product of Rev exon 2 (amino acids 31 to 165). (A) Amino acid sequence of the product of exon 2 of MA-1 Rev (4) and amino acid sequences encoded by cDNAs isolated from MA-1-infected ED cells (ME) and MA-1-infected horse macrophage cultures (HMC) (MM). (B) Amino acid sequences of products of cDNAs isolated from Th-1-infected HMC (A22, B11, F22, H21) and viral DNAs isolated from an EIAV-positive horse at the first and sixth febrile cycles (Th-1 and Th-6, respectively) (1). Missing sequences are due to the use of an internal 5′ primer for PCR amplification. (C) Amino acid sequences deduced from in vivo Rev exon 2 sequences obtained from GenBank (accession no. X63059, X16988, M14855, M18385, M18386, M18387, M18388, M87580, and M93674).

FIG. 3

In vitro assays of EIAV Rev activity. Transfection experiments were performed as described in the text. Two days posttransfection cells were harvested and lysates were normalized for the CAT reactions by a beta-galactosidase assay. Individual experiments included triplicate wells, and the data shown represents the means of at least three separate experiments. Error bars denote the standard errors of the means for all experiments. (A) Rev can trans activate through two discrete regions of EIAV. pcMARev transactivation of CAT reporter plasmids pERRE-All, pERRE-1, pERRE-2, and pDM138 is shown. Each reporter plasmid contains the EIAV sequences shown in Fig. 1B; pDM138 is the background reporter plasmid. Transfections and CAT assays were performed as described in the text, except that lysates from wells with pERRE-All were diluted fivefold to allow for the comparison. (B) Transactivation of pDM138 CAT reporter plasmid pERRE-All by EIAV Rev variants showing that amino acid variation in Rev alters the biological activity of the protein. (C and D) Transactivation of pDM138 CAT reporter plasmids pERRE-1 (C) and pERRE-2 (D) by EIAV Rev variants indicating that the full effects of variation require both RRE regions. CAT assays were performed for each experiment under conditions that resulted in approximately 20% acetylation for the pcMARev lysates. Therefore, although the activities of the reporter plasmids differ, all experiments appear on the same scale.

FIG. 4

Amino acid variation does not alter Rev-dependent alternative splicing. Rev-defective cells were transfected with 9 μg of variant-Rev plasmids. Total RNA was isolated and reverse transcribed with random hexamer primers. cDNA was amplified by PCR with EIAV primers specific for exon 1 and exon 4 by using a 5′ primer that was end-labeled with ³²P. PCR products were isolated and separated by electrophoreses through a denaturing 5% polyacrylamide gel. The locations of EIAV splicing products are shown. As a control, mRNAs from transfected plasmids were amplified with pCR3-specific primers flanking the Rev insert.