Synergistic stimulation of HIV-1 Rev-dependent export of unspliced mRNA tothe cytoplasm by hnRNP A1

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transpor t of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev re sponse element). However, the RRE does not permit Rev to stimulate the expo rt of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene in stability (INS) element contains RNA elements that can complement Rev activ ity. In the presence of the INS element and the RRE, Rev permits up to 30% of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' en d of the p17 gag gene INS element (5' INS) is functional and permits the ex port to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre -mRNA. Gel mobility shift assays and UV cross-linking experiments have show n that heterogeneous nuclear ribonucleoprotein (hnRNP) Al and a cellular RN A-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP Al and 50 kDa protein binding are inactive in the t ransport assay. To confirm that the hnRNP Al complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP Al was also ana lysed. When the high affinity hnRNP Al binding site was inserted into the P -globin reporter, Rev was able to increase the cytoplasmic levels of unspli ced mRNAs to 14%. In contrast, the mutant hnRNP Al binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport . We conclude that hnRNP Al is able to direct unspliced globin pre-mRNA int o a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm. (C) 1999 Academic Press.