No abstract

Recombinant Rev protein of human immuno- (20) have reported purification of recombinant HIV-1 Rev by using ion-exchange and gelfiltration chromatography, also under denaturing conditions, followed by protein refolding. While refolded Rev was shown to bind RRE RNA in vitro, no analysis ofthe protein structure was performed.We have developed an alternative method of purifying recombinant HIV-1 Rev from Escherichia coli that maintains the protein in a stable homogeneous form suitable for structural analysis. The purified protein binds specifically to RNA containing the RRE. Circular dichroism and intrinsic fluorescence spectroscopy indicate that the purified protein is structured. Protein characterization by gel-filtration chromatography and SDS/PAGE suggest that HIV-1 Rev forms a stable tetramer.EXPERIMENTAL PROCEDURES Cloning and Expression of HIV-1 rev. A synthetic rev gene segment for the first 56 amino acids of HIV-1 Rev (strain HXB2) was prepared from oligonucleotides; codons frequently used in E. coli were used (25). This gene segment was fused into the BamHI/Pvu II fragment from a cDNA clone of the HXB3 strain to produce a hybrid Rev protein (21). Expression of the rev gene was under the control of the APL promoter and was induced in midlogarithmic phase culture by temperature shift to 420C. After induction, the cells were allowed to accumulate HIV-1 Rev for 4 hr prior to harvesting by centrifugation. Cells were stored at -800C until needed for purification.Purification of Recombinant HIV-1 Rev. E. coli cells (400 g, wet weight) expressing HIV-1 Rev were suspended in 50 mM Tris HCl, pH 8.0/1 mM EDTA/0.5 mM phenylmethylsulfonyl fluoride (buffer A) at 0.25 g/ml and disrupted by sonication or by passing through a Manton-Gaulin homogenizer. Insoluble material was removed by centrifugation at 15,000 X g for 30 min. The soluble fraction was diluted 6-fold with buffer A containing 0.48 M NaCl and was applied onto a Q-Sepharose column (4.4 x 20 cm; Pharmacia) equilibrated with buffer A containing 0.4 M NaCl. The majority of cellular Abbreviations: HIV-1, human immunodeficiency virus type 1; RRE, Rev-responsive element. tTo whom reprint requests should be addressed. 7593The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

No abstract

The transcriptional activator protein (Tax) from human T-cell leukemia virus type 1 was expressed in yeast using several different promoters in several strains: In all instances, expression of Tax resulted in very strong aggregation of the yeast cells. This phenotype appears to be identical by all criteria tested to the flocculation phenotype of the dominant mutation flo 1. Of most significance, mutations in Tax that affect transactivation of the IL-2R alpha regulatory sequences, but retain their ability to activate the viral long terminal repeat also fail to yield the aggregation phenotype. Based on these findings, expression of Tax in yeast may prove to be a simple primordial system for examining the regulatory mechanisms and cellular functions involved in regulation of gene expression.

No abstract