Communicated by Fotis C. Kafatos, December 6, 1988 (received for review October 6, 1988) ABSTRACT rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag-and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named revresponsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.

The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic ␣-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128 -366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360 -366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo-and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.The nuclear receptor superfamily comprises a large set of ligand-regulated transcription factors. This superfamily includes receptors for steroid hormones, retinoids, thyroid hormone, and vitamin D 3 , as well as a large number of structurally and functionally related transcription regulatory proteins whose natural ligands are not yet known, the so-called orphan receptors (reviewed in Refs. 1 and 2). Nuclear receptors exhibit a modular structure with six distinct regions (referred to as regions A-F), which correspond to functional domains. The N-terminal region A/B is highly variable among nuclear receptors and contains a ligand-independent transactivation function AF-1 (2). Region C contains a highly conserved DNA binding domain (DBD) 1 composed of two zinc-coordinated modules and is responsible for specific binding to cognate response elements (Refs. 1 and 2 and references therein). The exact functions of regions D and F are not clear, although they appear to be well conserved for each receptor across species. Region D is postulated to function as a flexible hinge between the DBD and the ligand-binding domain (LBD), allowing rotational differences between these domains when dimeric receptors bind to direct, inverted, or palindromic repeats (2). Interestingly, the D regions of the thyroid hormone (TR) and retinoic acid receptors (RARs) interact with ...

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trslart) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels ofgag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.

The N-termnal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pue-chase experiments showed that the depletion of intracellular apoB-17follows an apparent first-order kinetics with tl2 = 51 min.Under conditions of continuous labeling, >60% of the total synthesized apoB-17 was secreted in a soluble form, =98% lipid-poor and -2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5-and 2.5-fold increases in the amount of labeled apoB-17 in the p < 1.063 g/ml and 1.063 < p < 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p > 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/ apoB-17, 7:1 (wt/wt), are 239 ± 43 A in diameter and 61 ± 4 A thick and contain -2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% a-helix, 36% 13-sheet, 9% (3-turn, and 16% random coil.To be consistent with this model >70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B‐100 (apoB‐100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5′ and 3′ untranslated regions which are 128 and 301 nucleotides respectively. The DNA‐derived protein sequence shows that apoB‐100 is 513,000 daltons and contains 4560 amino acids including a 24‐amino‐acid‐long signal peptide. The mol. wt of apoB‐100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non‐amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid‐binding domains of apoB‐100. The protein contains 25 cysteines and 20 potential N‐glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor‐binding domain of apoB‐100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15‐kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB‐100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.

The intestinal effects of Clostridium difficile toxin A are initiated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (

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