Mutagenesis of EIAV TAT reveals structural features essential for transcriptional activation and TAR element recognition.
Certain members of the lentivirus subfamily of retroviruses encode unique transcriptional activator (Tat) proteins that modify the transcription complex after binding to the 5' end of nascent viral mRNA. The Tat proteins are modular, containing RNA-binding and activation domains that can be exchanged between different Tat proteins or replaced with heterologous protein fragments. While there is considerable sequence conservation among the divergent Tat proteins, there are also some structural differences that might be informative. For example, a cluster of basic amino acids in HIV-1 Tat is sufficient for RNA binding in vivo and in vitro. The homologous region of EIAV Tat is necessary but not sufficient for recognition of its cognate cis-acting RNA element; the entire C-terminal 26 amino acids of EIAV Tat, including the basic patch, are required. To better understand the structure-function relationships in EIAV Tat, we have generated a battery of expression plasmids encoding insertion, deletion, and missense mutations in the carboxy-terminal region of the tat gene. The plasmids were tested for their ability to trans-activate the EIAV promoter or to trans-inhibit a heterologous Tat protein. A mutation of a glutamine to an arginine in the cluster of basic residues generated a potent trans-dominant inhibitor of both EIAV and HIV-1 Tat, indicating that the mutation abolished RNA binding but did not alter the activation domain. Mutations at the extreme C-terminus of EIAV Tat impaired both RNA binding and activation domain functions, suggesting effects on secondary or tertiary structure.
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Laboratory of Viral Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Newbold
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D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics* / Q000276:immunology; D015497:HIV-1 / Q000235:genetics; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus