Uses and mechanism of the woodchuck hepatitis virus posttranscriptional regulatory element in the enhancement of gene expression [Utilisations et mécanisme de l'élément de régulation posttranscriptionel du virus de l'hépatite de la marmotte américaine dans la mise en valeur de l'expression des gènes].

Résumé

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is a cis-acting RNA sequence which evolved to enhance the expression of intronless viral messages. The WPRE is composed of three modular subelements, each contributing to its ability to localize unspliced messages to the cytoplasm. To determine whether the ability of the WPRE to enhance expression could be useful in non-viral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of 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) to cell lines or to primary fibroblasts. To elucidate the mechanism of enhancement, we analysed nuclear and cytoplasmic RNA fractions of transduced or stably transfected cell lines. We find that the presence of the WPRE causes a significant increase in the levels of GFP RNA detected in both cellular compartments, independent of copy number. Analysis of RNA reveals an increase in poly(A) tail length to the physiological maximum on messages bearing the WPRE. This increase is established in the nucleus. We compared the tail lengths of messages containing the WPRE and other characterized viral export elements, revealing that the WPRE is unique both in its ability to maximize poly(A) tail length and in its ability to enhance expression. The WPRE maximizes poly(A) tail length independently of the identity of the polyadenylation signal or promoter. In addition to increasing the maximum tail length on a given message, the WPRE protects messages from deadenylation in the cytoplasm. Mutational analysis of the WPRE indicates a correlation of maximized and maintained poly(A) tail length with the enhancement of expression conferred by the WPRE, and assigns these functions to different cis-acting sequences within the WPRE. The chain-terminating adenosine analog cordycepin specifically antagonizes the effect of the WPRE on cytoplasmic poly(A) tail length, suggesting that the WPRE facilitates continued polyadenylation. These results suggest that the WPRE positively affects gene expression in the nuclear and cytoplasmic compartments by influencing the 3’ metabolism of messages.