K. Verhoef et al., DETERMINATION OF THE MINIMAL AMOUNT OF TAT ACTIVITY REQUIRED FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION, Virology, 237(2), 1997, pp. 228-236
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.