Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy.

Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase.

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R. , Taagepera, S., Schwartz, M. & Wang, J. (1996) Proc. Natl. Acad. Sci. USA 93, 15174-15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevDelta3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(-), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(-) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element.

The hepatitis B virus posttranscriptional regulatory element (HBVPRE) is a cis-acting RNA element that partially overlaps with enhancer I and is required for the cytoplasmic accumulation of HBV surface RNAs. We find that the closely related woodchuck hepatitis virus (WHV), which has been shown to lack a functional enhancer I, also contains a posttranscriptional regulatory element (WPRE). Deletion analysis suggests that the WPRE consists of three independent subelements. Comparison of the bipartite HBVPRE and tripartite WPRE activities reveals that the tripartite WPRE is two to three times more active than the bipartite HBVPRE. Mutation of a single WPRE subelement decreases WPRE activity to the level of the HBVPRE. Bipartite and tripartite chimeras of the WPRE and HBVPRE possess activities which suggest that elements containing three subelements are posttranscriptionally stronger than those containing two. These data demonstrate that the posttranscriptional regulatory element is conserved within the mammalian hepadnaviruses and that its strength is determined by the number of subelements within the RNA.

The role of turns in the structure of an alpha-helical protein.

The turns joining segments of secondary structure have been proposed to be key elements in dictating the folded structures of native proteins. An alternative view assumes that turns play a passive role and are merely default structures that occur as a consequence of interactions between antiparallel segments of secondary structure, with chain reversal being dictated by the context surrounding the turn and not by the sequence of the turn itself. The solvent-exposure of turns and their tolerance to evolutionary variance suggests that they may have little or no effect on the formation of native structures. Previous investigations have focused on various types of beta-turns that connect antiparallel beta-strands with comparatively little reported on the structural role of interhelical turns. Here we probe the structural importance of such a turn in an antiparallel 4-helix bundle by randomly substituting an interhelical tripeptide in cytochrome b-562 with many different amino-acid sequences. Thirty-one of the resulting substituted proteins were characterized and all of them were shown to fold into stable, native-like structures. These results suggest that this interhelical turn does not does not play a dominant role in determining the folded structure of this antiparallel 4-helix bundle.

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles.

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.

A membrane-bound protein kinase from mouse liver stimulated by iron.

At microM levels, iron stimulates strongly the in vitro phosphorylation of a membrane protein from mouse liver. This phosphorylation also occurs in the absence of other added divalent cations but to a lower degree. In SDS gels the phosphorylated protein has app. Mr 250 000 in the absence of mercaptoethanol and Mr 130 000 in its presence. It is phosphorylated on threonine residues.

Purification and characterization of the cytoplasmic casein kinase I from rat liver.

Two cyclic nucleotide-independent protein kinases, which preferentially utilize casein and phosvitin as substrates, exist in rat liver. In contrast to cytosol the "light' form of these enzymes was predominant in the "microsomal extract'. This form (30 000-40 000 daltons, casein kinase I) was separated from the "heavy' form (130 000 daltons, casein kinase II) by gel filtration. This enzyme was then purified by successive chromatography on carboxymethyl-Sephadex, phosvitin-Sepharose and hydroxyapatite. The activity of the purified enzyme was 2000-3000-fold the casein kinase activity of the cytosol. It had a s20,w of approx. 3 S as determined on sucrose density gradient. After iodination or incubation with [gamma- 32P]ATP, it was analyzed on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and appeared to be composed of a single polypeptide (36 000 +/- 1000 daltons) which self-phosphorylated. In contrast to casein kinase II, casein kinase I preferentially utilized ATP over GTP. The Km value for ATP was determined to be 14 micro M. The Km value for phosvitin was 0.17 mg/ml. Casein kinase I phosphorylated sites different from those of casein kinase II (as shown with ribosomes or SV40 T antigen). Casein kinase I was further characterized by studying its thermal stability. The half-life at 37 degree C was 6 min and 1 min 30 s at 54 degree C. In the presence of two substrates (ATP and phosvitin), the half-life at 54 degree C increased from 1 min 30 s to 4 min. Hemin strongly increased the rate of inactivation of the casein kinase I at 37 degree C in the absence or presence of the substrates. N-ethylmaleimide also inactivated casein kinase I. Phosvitin, though not ATP, protected the enzyme. This observation may indicate that thiols are involved at the binding site of the enzyme for the protein substrate.

Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.