Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation.
The Tat protein coded by HIV-1 is a unique eukaryotic transactivator. It activates gene expression from the viral LTR by its interaction with a nascent RNA element (TAR) located at the 5' end of all HIV-1 transcripts. Tat appears to bind to its target RNA structure in a highly sequence-specific manner. The TAR-binding activity of Tat has been localized in an Arg-rich basic domain located between residues 49 and 57 of the Tat protein. We have carried out domain substitution studies with heterologous basic domains which are also implicated in RNA binding. Here, we report that a 19 or a 12 amino acid region from the N-terminus of HTLV-I Rex can functionally substitute for the Tat basic domain. In contrast, the Arg-rich domains of the N gene products of bacteriophages lambda and 21 do not functionally substitute for the Tat basic domain. The positive and negative effects of various domain substitution mutants have facilitated identification of a consensus sequence (Arg/Lys-X-X-Arg-Arg-X-Arg-Arg) in the basic domain required for Tat activity. Conversion of the functionally inactive basic domain of the lambda N protein to the consensus motif restored the transactivation function of the Tat-N chimeric protein. Similarly, the Rex basic domain containing scrambled sequences unrelated or partially related to the consensus motif were either totally defective in transactivation or exhibited reduced activity. Our results further suggest that the activity of the core Arg motif may be enhanced by the presence of Gln or Asn within the basic domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Subramanian
T
T
Institute for Molecular Virology, St Louis University Medical Center, MO 63110.
Govindarajan
R
R
Chinnadurai
G
G
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D002460:Cell Line; D002678:Chimera; D005455:Fluorescent Antibody Technique; D016370:Gene Products, rex / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics*; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015533:Transcriptional Activation; D054322:tat Gene Products, Human Immunodeficiency Virus
D016370:Gene Products, rex; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D001120:Arginine