DETERMINATION OF THE MINIMAL AMOUNT OF TAT ACTIVITY REQUIRED FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a po tent trans-activator of transcription from the viral LTR promoter. Pre vious mutagenesis studies have identified domains within Tat responsib le for binding to its TAR RNA target and for transcriptional activatio n. The minimal Tat activation domain is composed of the N-terminal 48 residues, and mutational analyses identified a cluster of critical cys teines. The importance of four highly conserved aromatic amino acids w ithin the activation domain has not been thoroughly investigated. We h ave systematically substituted these aromatic residues (Y26, F32, F33, Y47) of the HIV-1 LAI Tat protein with other aromatic residues (conse rvative mutation) or alanine (nonconservative mutation). The activity of the mutant Tat constructs was measured in different cell lines by t ransfection with a LTR-CAT reporter plasmid. The range of transcriptio nal activities measured for this set of Tat mutants allowed careful as sessment of the level of Tat activity required for optimal viral repli cation. To test this, the mutant Tat genes were introduced into the pL AI infectious molecular clone and tested for their effect on virus rep lication in a T-cell line. We found that a twofold reduction in Tat ac tivity already affects viral replication, and no virus replication was measured for Tat mutants with less than 15% activity. This strict cor relation between Tar activity and viral replication demonstrates the i mportance of the Tat function to viral fitness. Interestingly, a less pronounced replication defect was observed in primary cell types. This finding may correlate with the frequent detection of proviruses with Tat-inactivating mutations in clinical samples. (C) 1997 Academic Pres s.