A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat.

Abstract

Regulation of human immunodeficiency virus (HIV) gene expression is dependent on specific regulatory regions in the long terminal repeat. These regions include the enhancer, SP1, "TATA," and trans-activating (TAR) regions. In addition, viral regulatory proteins such as tat and rev are important in regulating HIV gene expression. The mechanism of tat activation remains the subject of investigation, but effects at both transcriptional and posttranscriptional levels seem likely. Previous mutagenesis of the tat protein revealed that the amino terminus, the cysteine-rich domain, and the basic domain were all required for complete tat activation. Mutants of other viral trans-acting regulatory proteins, including ElA, tax, and VM65, have been identified that were capable of antagonizing the activity of their corresponding wild-type proteins. We wished to determine whether mutants of the tat protein could be identified that exhibited a similar phenotype.

A Mutant Tat Protein Provides Strong Protection from HIV-1 Infection in Human CD4+T Cells

“…Like similarly mutated one-exon Tat mutants, Nullbasic exhibited dominant negative effects on Tat-dependent HIV-1 gene expression. However, unlike previously reported mutants (Green et al, 1989;Pearson et al, 1990;Modesti et al, 1991;Ulich et al, 1996), Nullbasic inhibited reverse transcription and also effectively suppressed the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity (Meredith et al, 2009). HeLa CD4 + cells constitutively expressing Nullbasic were strongly protected from a spreading infection by HIV-1 (Meredith et al, 2009).…”

“…Mutant forms of Tat that inhibit HIV-1 gene expression in T cell lines have been described but these proteins were truncated within the basic domain (amino acids 49-57), expressed only the first exon, or were truncated in the second exon (86-amino acid Tat) and had fewer substitutions in the basic domain (Pearson et al, 1990;Modesti et al, 1991). Some of these mutant Tat proteins were shown to provide modest protection from HIV-1 replication when expressed in human T cell lines (Ulich et al, 1996).…”

“…Dominant negatives for many viral proteins have been described including Tat (Green et al, 1989;Pearson et al, 1990;Modesti et al, 1991), Rev (Bevec et al, 1992;Malim et al, 1992), Vif (Walker et al, 2010), Nef (Fackler et al, 2001), and Gag (Trono et al, 1989;Furuta et al, 1997;Lee et al, 2009;Checkley et al, 2010). For example, engineered Tat proteins with altered basic domains possess trans-dominant negative phenotypes against wild-type Tat function in transcription (Pearson et al, 1990), and a trans-dominant-negative Rev mutant called RevM10 has been described that was shown to inhibit wildtype Rev function (Malim et al, 1991). The RevM10 mutant protein is dominant negative because it retains the ability to bind the HIV-1 Rev response element (RRE) but is unable to promote CRM1 (chromosome region maintenance 1)-mediated export of the viral mRNA from the nucleus, thereby inhibiting HIV-1 replication (Malim et al, 1991;Stauber et al, 1998).…”

HIV-1 proprotein processing as a target for gene therapy

“…To date, several RNA and protein agents have been introduced into susceptible cells and tested as inhibitors of HIV-1 replication. These studies have included dominant negative mutants of HIV structural (Gag, 1 Env 2 ) or regulatory (Rev, 3 Tat, 4 PR 5 ) proteins, TAR 6 and RRE 7 RNA decoys molecules for the viral proteins (Tat, REV), antisense RNAs, 8 antibodies directed against Rev, 9 gp120 10 or Tat, 11 and ribozyme designed to cleave at different conserved sites of the HIV-1 genome. 12,13 These approaches all target phases of HIV-1 replication before virus assembly.…”

Isolation of efficient antivirals: genetic suppressor elements against HIV-1

“…A cluster of sense-oriented GSEs were isolated from the second exon of the rev and tat genes, a sequence contained in both Tat and Rev transdominants. 21,22 This sequence also overlaps with the sequence of the envelope. A GSE from the rev/tat sense cluster (IGX-230) showed a comparable profile in productive infection as the transdominant, RevM10 (Figure 4a).…”