Mutations in the Nuclear Export Signal of Human Ran-binding Protein RanBP1 Block the Rev-mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1

Abstract

We identified a region in the human Ran GTPasebinding protein RanBP1 that shares similarities to the nuclear export signal of the inhibitor of the cAMP-dependent protein kinase. Mutational analysis confirmed that this region is responsible for the cytoplasmic accumulation of RanBP1 and can functionally replace the nuclear export signal of Rev of human immunodeficiency virus type 1. We showed that RanBP1 interferes with Rev-mediated expression of human immunodeficiency virus type 1, whereas the RanBP1 with inactivated nuclear export signal abrogates Rev function. Expression of a Rev-independent molecular clone, which is regulated via the constitutive transport element (CTE) of the simian retrovirus type 1, is not affected. These findings indicate that Rev and RanBP1 compete for the same nuclear export pathway, whereas Rev-and the CTE-mediated pathways are distinct. The inhibition of Rev function is independent of the ability of RanBP1 to associate with Ran and therefore, it is not likely a result of interference with Ran function. These data suggest that RanBP1 interacts with Rev at the putative nuclear receptor and, hence, shares a step in posttranscriptional pathway with Rev.One of the best studied examples of regulation at the posttranscriptional level is that of the Rev-responsive element (RRE) 1 -containing mRNAs of HIV-1 (for reviews, see Refs. 1-5). The Rev protein binds to the RRE and promotes the export of the RRE-containing mRNA to the cytoplasm and their expression. Rev contains an RNA-binding domain and a nuclear export signal (NES). Mutations in the NES are transdominant (TD) and lead to inhibition of Rev's nucleocytoplasmic export resulting in inhibition of HIV expression. Nucleoporin-like proteins hRip/Rab have been shown to interact with the NES of Rev and are believed to be involved in the nuclear export of Rev (6, 7). Similar NES elements have been identified in the inhibitor of cAMP-dependent protein kinase (PKI) (8) and the 5 S RNA-binding protein TFIIIA (9) and have been shown to functionally replace the NES of HIV-1 Rev. We identified a related NES region in the Ran GTPase-binding protein 1 of human (hRanBP1) (10, 11) and mouse RanBP1/ HTF9-A (mRanBP1) (12) origin. They belong to a family of proteins that bind to the GTP-bound Ran that includes known and putative nucleoporins as well as proteins of unknown function (13-19). Ran GTPase is required for active transport of macromolecules through the nuclear pore (for recent reviews, see Refs. 20 and 21). Here, we show that the NES signals of RanBP1 and Rev functionally cross-interfere, indicating a competition for a common export pathway. NES(Ϫ)RanBP1 mutants completely abrogate Rev-mediated regulation of HIV-1 without affecting the regulation mediated by the posttranscriptional control element (CTE) of simian retrovirus type 1. Taken together, our data suggest that RanBP1 and Rev share a nuclear export pathway, which is distinct from the CTE-mediated pathway. EXPERIMENTAL PROCEDURESRecombinant DNA and Transfections-HIV-1 molecular clone pNL4...

Isolated Mammalian andSchizosaccharomyces pombeRan-binding Domains RescueS. pombe sbp1(RanBP1) Genomic Mutants

“…Each of these proteins was subcloned with a T7 epitope tag in frame at its amino terminus (see MATERIALS AND METH-ODS). Consistent with previous studies (Richards et al, 1996;Zolotukhin and Felber, 1997), cells transfected with a construct expressing T7-tagged fulllength mouse RanBP1 showed largely cytoplasmic staining (Figure 2, left panels). In contrast, in cells expressing the T7-tagged mouse RBD, which lacks the NES motif (Figure 1), staining was largely nuclear (Figure 2, left panels).…”

“…This localization appears to be an active process, because the isolated RBD of RanBP1 localizes predominantly to the nucleus and the presence of a leucinerich nuclear export signal (NES) motif in the carboxylterminal portion of the full-length protein is required for its cytosolic localization (Richards et al, 1996;Zolotukhin and Felber, 1997). It is unclear whether the NES mediates normal shuttling of RanBP1 between nuclear and cytosolic compartments or whether it serves to ensure confinement of RanBP1 to the cytosol.…”

“…Human RanBP1 proteins in which single leucine or valine residues within the carboxyl-terminal NES are replaced by alanine accumulate in the cell nucleus (Richards et al, 1996;Zolotukhin and Felber, 1997), suggesting that RanBP1 can readily enter the cell nucleus, and that an efficient export mechanism is required to maintain its predominantly cytoplasmic localization. The localization of mammalian RanGAP/ yeast Rna1p to the cytoplasm appears likewise to be dynamic.…”

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates

“…To determine the specificity of the interaction between TAP and the selected proteins, we performed in vitro binding assays+ [ 35 S]methionine-labeled putative TAP partners were synthesized in vitro in rabbit reticulocyte lysates and assayed for binding to glutathione agarose beads coated with either GST-TAP, GST alone, or various TAP fragments fused to GST+ Binding of RanBP2, p205, and KIAA0169 was not further investigated+ In preliminary experiments we noticed that some of the selected proteins bound to GST-TAP only in the presence of nuclear extracts+ This was the case for CRM1, importin-b, Nup88, Nup93, and RanGAP1 (data not shown)+ Additional experiments indicate that TAP/CRM1 interaction was bridged by CAN and probably by other nucleoporins present in the column (data not shown)+ Because TAP-mediated export is distinct from the CRM1 export pathway (Pasquinelli et al+, 1997;Saavedra et al+, 1997;Zolotukhin & Felber, 1997;Bogerd et al+, 1998;Otero et al+, 1998), we conclude that TAP/CRM1 interaction is not relevant for TAP function+ However, our results indicate that the two proteins share common binding sites at the NPC (see below)+ Binding of in vitro-synthesized transportin, CAN, Nup98, hGle2, and E1B-AP5 to GST-TAP neither required nor was stimulated by addition of nuclear extracts (Figs+ 3, 4, and 5 and data not shown) suggesting that these interactions may be direct, and thus were characterized further+ When GST pull-down assays were performed with various fragments of TAP, hGle2 interacted with the C-terminal domain of TAP (TAP371-619) but not with its N-terminal domain (TAP1-372) (Fig+ 3A, lanes 3-5)+ The C-terminal domain of TAP also interacted with all nucleoporins analyzed in this study (Fig+ 7)+ In contrast, E1B-AP5 associates with the N-terminus (TAP1-372) but not with the C-terminal domain of TAP (TAP371-619; Fig+ 3B, lanes 4 and 5)+ Shorter N-terminal TAP fragments (TAP1-265 and TAP1-185) exhibited a reduced binding affinity for E1B-AP5 (Fig+ 3B, lanes 6 and 7), whereas deletion of TAP's first 60 amino acids severely impaired its binding to E1B-AP5 (TAP61-372; Fig+ 3B, lane 8)+ This observation was supported further by the GST pull-down assay shown in Figure 3C+ In this assay, various TAP deletion mutants synthesized in vitro were tested for their ability to bind to an E1B-AP5 fragment expressed in E. coli as a GST fusion+ This E1B-AP5 fragment (101-619) binds TAP with the same efficiency as the full-length protein (data not shown)+ Deletion of TAP residues 22-55 severely impaired its binding to E1B-AP5 (Fig+ 3C, lanes 4-6), whereas short deletions downstream of position 55 had no effect+ Together, our results indicate that fragment 1-372 represents the E1B-AP5 binding domain of TAP+ Within this domain, the first 55 amino acids appear to contribute substantially to the affinity of the interaction+ Although we can- not rule out that the interaction of TAP with hGle2 and E1B-AP5 is mediated by RNA or by a factor present in the reticulocyte lysate, we consider this possibility unlikely as these interactions were neither prevented by microccocal nuclease treatment nor stimulated by including increasing amounts of HeLa extracts, reticulocyte lysate, or RNA in the binding reactions (data not shown and Fig+ 3D)+ Because the E1B-AP5-binding domain of TAP overlaps with its CTE-binding domain, we next tested the effect of the CTE RNA on TAP/E1B-AP5 interaction+ Full-length TAP and an alanine scan mutant (TAP295) that no longer interacts with the CTE RNA …”

RNA-binding Motif Protein 15 Binds to the RNA Transport Element RTE and Provides a Direct Link to the NXF1 Export Pathway

“…Recombinant DNA-The reporter plasmids pNLgag, pNLCgag, or pDM138 containing RTE, mutant RTE, CTE, or RRE (34 -38), pDM128/PL and pDM128/B (39), the CMVgag/pol plasmids containing a polylinker or the MPMV-CTE (40), the expression plasmids for NXF1 (3), p15/NXT1 (41), HIV-1 rev (pCMVsrev) (42), pN-NXF1 and pN-Rev (39), luciferase (RSVluc) (43), GFP (pFRED25 and pFRED143) (44), secreted alkaline phosphate (SEAP) (45), and UAP56 (46) have been described. pNLgagRTE(IAP23L92) contains the RTE from the active retroelement IAP92L23 (47).…”