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. It has been shown that the basic domain is required for targeting Tat to the nucleus and nucleolus. The chimeric Tat proteins containing the basic domains of Rex and the N proteins are also localized in the nuclear/nucleolar region. Since the functionally inactive Tat-N chimeric proteins are still efficiently targeted to the nuclear/nucleolar compartments, our results suggest that nuclear/nucleolar localization alone is not sufficient for transactivation and demonstrate a direct role for the Arg motif in Tat function other than that required for nuclear/nucleolar localization.