Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.
We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.
Stephens
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RM
Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21701.
Derse
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D005091:Exons; D015723:Gene Library; D015694:Gene Products, rev / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics*; D006736:Horses; D007106:Immune Sera; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D007668:Kidney; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012367:RNA, Viral / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004279:DNA, Viral; D015694:Gene Products, rev; D015696:Gene Products, tat; D007106:Immune Sera; D010455:Peptides; D012367:RNA, Viral; D015534:Trans-Activators