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 potent trans-activator of transcription from the viral LTR promoter. Previous mutagenesis studies have identified domains within Tat responsible for binding to its TAR RNA target and for transcriptional activation. The minimal Tat activation domain is composed of the N-terminal 48 residues, and mutational analyses identified a cluster of critical cysteines. The importance of four highly conserved aromatic amino acids within the activation domain has not been thoroughly investigated. We have systematically substituted these aromatic residues (Y26, F32, F38, Y47) of the HIV-1 LAI Tat protein with other aromatic residues (conservative mutation) or alanine (nonconservative mutation). The activity of the mutant Tat constructs was measured in different cell lines by transfection with a LTR-CAT reporter plasmid. The range of transcriptional activities measured for this set of Tat mutants allowed careful assessment of the level of Tat activity required for optimal viral replication. To test this, the mutant Tat genes were introduced into the pLAI infectious molecular clone and tested for their effect on virus replication in a T-cell line. We found that a twofold reduction in Tat activity already affects viral replication, and no virus replication was measured for Tat mutants with less than 15% activity. This strict correlation between Tat activity and viral replication demonstrates the importance 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.
Verhoef
K
K
Department of Human Retrovirology, University of Amsterdam, Academic Medical Center, Amsterdam, 1100 DE, The Netherlands.
Koper
M
M
Berkhout
B
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D015696:Gene Products, tat / Q000502:physiology*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus