Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis.

Abstract

Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptormediated pathway.Human immunodeficiency virus (HIV) gene expression is tightly controlled by numerous cellular (1, 2) and viral (3-6) trans-acting regulatory factors. The HIV genome alone encodes at least three trans-acting regulatory proteins not present in other animal retroviruses (7-11). It is well established that expression of two of these proteins, referred to as Tat (7) and Rev [regulator of virion expression, previously referred to as art or trs (11)], is required for virus replication (12-15). The tat gene encodes a nucleolar protein (16, 17) that is a positive regulator of virion gene expression. The cisacting control elements responsive to Tat, referred to as TAR (18,19), are present between nucleotides -17 to +44 (18, 19) in the long terminal repeat (LTR) and are thus present in the 5' untranslated leader region of all viral messages. Mechanisms operating at both the transcriptional (18,(20)(21)(22)(23) and posttranscriptional (20,24,25) level have been proposed to explain tat function.The Tat protein contains at least three functional domains (17): (i) a negatively charged region at the amino terminus, (ii) a cysteine-rich region, and (iii) a highly basic region. Studies of tat mutants indicate that the amino-terminal residues and cysteines 22, 25, 27, and 37 are required for Tat function (17). The cysteine-rich region has been proposed to form a metal binding "finger" common to several transcriptional regulatory proteins. Frankel et al. have proposed an alternative structure whereby the cysteines are involved in the formation of a metal-linked dimer (26). The presence of both the highly charged region and the cysteine-rich cluster has hampered protein purification efforts.In the present study we describe a simple and efficient method for purification of biologically active Tat protein.

Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids.

“…Formation of protein recognition sites on DNA can also proceed through a similar mechanism. For example, some DNAbinding proteins {i.e., Fos and Jun) form protein dimers through a so-called leucine zipper, which then creates a DNA-binding site that lies adjacent to the dimerization domain {Gentz et al 1989a;Landschulz et al 1988). The mechanism for Rev-mediated export of viral structural mRNAs from the nucleus to cytoplasm is not understood.…”

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

“…IB) (Gentz et al 1989). The wildtype Tat protein is biologically active, as demonstrated by both scrape-loading and simple addition of Tat to the culture medium (Gentz et al 1989). Furthermore, this protein specifically binds to the bulge of the HIV-1 TAR with high affinity (Roy et al 1990).…”

“…Tat protein, expressed in Escherichia coli as a fusion protein with 6 histidine residues and highly purified by affinity chromatography, was used in this experiment (Fig. IB) (Gentz et al 1989). The wildtype Tat protein is biologically active, as demonstrated by both scrape-loading and simple addition of Tat to the culture medium (Gentz et al 1989).…”

“…The expression of the histidine-tailed Tat proteins in E. coli and the purification through nickel-agarose chromatography were performed as described (Gentz et al 1989), except that 50 mM imidazole was used for elution instead of low pH. Tat proteins were further treated and renatured.…”

I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention.

“…Expression of IKB/MAD-3 in Escherichia coli was carried out by cloning the IKB/MAD-3 eDNA into the pDS expression plasmid (Gentz et al 1989). Expression and purification of IKB/MAD-3 were carried out by a protocol described previously (Ruben et al 1992b).…”