The life cycle of the human immunodeficiency virus (HIV) and other retroviruses is dependent upon nuclear transport. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA that must enter the nucleus. The viral regulatory proteins, Rev and Tat, have been identified as key modulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We are examining the processes of nuclear protein import and export both in vivo and in vitro using the HIV Rev protein. The Rev protein actively shuttles between nucleolus and cytoplasm and mediates premature export of unspliced retroviral RNAs. Using digitonin permeabilized cultured cells, we developed an in vitro assay for studying both nuclear import and export as well as nucleolar accumulation of molecules such as HIV Rev. These data suggest that dissociation from the nucleolus is an ATP-dependent process. Utilizing Fluorescence Recovery After Photobleaching, Rev was found to be immobilized at the nucleolus, further strengthening the role for an ATP-dependent release from the nucleolus. This regulated nucleolar localization may reflect a more widespread phenomenon since it has been observed for other nucleolar proteins involved in apoptosis and cell cycle control. The system we have described should allow for the identification of the requirements for regulated nucleolar targeting, as well as nuclear transport of other shuttling proteins. We are currently optimizing this system for use in a high throughput screen to mine for small molecule inhibitors of these key regulatory steps. We have recently demonstrated the leader peptide of MMTV Env precursor (MMTV-p14) is translocated to the nucleoli of murine lymphomas that harbor MMTV. Like Rev, p14 contains nuclear/nucleolar targeting signals. Furthermore both Rev and MMTV-p14 bind similar cellular targets (B23 and ribosomal protein L5). Based on the similar characteristics of these two proteins, MMTV-p14 may competitively impair the function of Rev, thus disrupting the HIV life cycle. The proposed use of the MMTV-p14 peptide has several advantages over previous dominant negative approaches using altered HIV gene products. The MMTV-p14 sequence is expected to have little effect on normal functioning in primates or humans. It also may avoid destruction mediated by immune reactions to HIV in infected cells. We are examining small molecule kinase inhibitors in high throughput screens to examine the localization of both HIV-Rev GFP and MMTV p14. The nuclear pore complex mediates all traffic between the nucleus and cytoplasm. This complex contains proteins that are highly decorated at serine and theronine residues by N-acetyl glucosamine. The exact role of this type of glycosylation is unclear. As it shares many of the characteristics of phosphorylation, it is thought to represent a type of signalling mechanism. Our laboratory has cloned and expressed the enzymes responsible for the addition (O-linked GlcNAc Transferase, OGT) and removal (O-GlcNAcase) of this sugar moiety. These enzymes are currently being used in conjunction with our in vivo and in vitro assay for nuclear transport (see above) to determine the exact role of the glycosylated nuclear pore complex in nuclear import and export.
