Mutational analysis of the equine infectious anemia virus Tat-responsive element.
A hairpinlike structure is predicted to exist at the 5' end of equine infectious anemia virus (EIAV) RNA which is similar in many ways to the human immunodeficiency type 1 (HIV-1) Tat-responsive element (TAR). In EIAV, this structure has a shorter stem than in HIV-1 and lacks the uridine bulge. Primer extension analysis of EIAV RNA was used to identify the transcriptional start site in the viral long terminal repeat. Premature termination of primer elongation at the predicted double-stranded RNA region was frequently observed and suggests that the inferred hairpin structure exists under these conditions. We have functionally characterized EIAV TAR by site-directed mutagenesis and transient gene expression analysis. It is demonstrated here that the secondary structure of this element is essential for Tat action. Mutations that disrupted base pairing abolished TAR function, and compensatory mutations that restored the stem structure resulted in Tat activation. The TAR loop appears to be closed by two U.G base pairs that are likely to provide a unique structural motif recognized by the Tat protein. With one exception, substitutions of nucleotides within the EIAV loop sequence decreased TAR function. All nucleotide substitutions of the cytidine at position +14 increased EIAV Tat responsiveness; however, its deletion abolished trans activation. Our results lead us to propose that the EIAV and HIV-1 Tat systems employ closely related cis- and trans-acting components that probably act by the same mechanism.
Carvalho
M
M
Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21702-1201.
Derse
D
D
D001483:Base Sequence; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics*; D006860:Hydrogen Bonding; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D012367:RNA, Viral / Q000235:genetics / Q000648:ultrastructure; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D015533:Transcriptional Activation
D015696:Gene Products, tat; D009841:Oligonucleotides; D012367:RNA, Viral