The adenovirus type 5 E1B-55K oncoprotein actively shuttles in virus-infected cells, whereas transport of E4orf6 is mediated by a CRM1-independent mechanism.

The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the "driving force" for adenoviral late mRNA export.

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Rapid evaluation and optimization of recombinant protein production using GFP tagging.

The isolation of recombinant proteins from bacterial or eukaryotic systems often requires a laborious optimization of expression and purification conditions. To greatly facilitate this procedure we included the green fluorescent protein (GFP) in bacterial expression vectors. This approach allowed us to sensitively detect the GFP hybrid proteins already in intact bacterial cells using a fluorescence microscope. To rapidly analyze a variety of conditions essential for protein expression, the GFP signal, indicative of expression levels, was directly quantitated in live bacterial suspensions using a fluorescence plate reader. Thus, GFP tagging not only allows one to directly monitor protein expression in general but also appears to increase protein stability or solubility.

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

Rab11b is essential for recycling of transferrin to the plasma membrane.

Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways. The Rab11 subfamily consists of two highly conserved members, Rab11a and Rab11b. Rab11a has been localized both to the pericentriolar recycling endosome and to the trans-Golgi network and functions in recycling of transferrin. However, the localization and function of Rab11b are completely unknown. In this study green fluorescent protein (GFP)-tagged Rab11b was used to determine its subcellular localization. GFP-Rab11b colocalized with internalized transferrin, and using different mutants of Rab11b, the role of this protein in transferrin uptake and recycling was examined. Two of these mutants, Rab11b-Q/L (constitutively active) and Rab11b-S/N (constitutively inactive), strongly inhibited the recycling of transferrin. Interestingly, both of them had no effect on transferrin uptake. In contrast, the C-terminally altered mutant Rab11b-DeltaC, which cannot be prenylated and therefore cannot interact with membranes, did not interfere with wild-type Rab11b function. From these data we concluded that functional Rab11b is essential for the transport of internalized transferrin from the recycling compartment to the plasma membrane.

Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity.

Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of the Herpesviridae family have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.

Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA.

Dendritic cells (DCs), nature's adjuvant, must mature to sensitize T cells. However, although the maturation process is essential, it is not yet fully understood at the molecular level. In this study, we investigated the course of expression of the unique hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A), which is part of a particular RNA nuclear export pathway, during in vitro generation of human DCs. We show that eIF-5A expression is significantly upregulated during DC maturation. Furthermore, an inhibitor of the hypusine modification, GC7 (N(1)-guanyl-1, 7-diaminoheptane), prevents CD83 surface expression by apparently interfering with nucleocytoplasmic translocation of the CD83 mRNA and, importantly, significantly inhibits DC-mediated T lymphocyte activation. The data presented suggest that CD83 mRNA is transported from the nucleus to the cytoplasm via a specific nuclear export pathway and that hypusine formation appears to be essential for the maturation of functional DCs. Therefore, pharmacological interference with hypusine formation may provide a new possibility to modulate DC function.

Human ribosomal protein L5 contains defined nuclear localization and export signals.

Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21-37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255-297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101-111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2.

The E1B-55K and E4orf6 oncoproteins of adenovirus type 5 are involved in the export of viral mRNAs. Previously, it was suggested that a complex composed of E1B-55K and E4orf6 serves as a nucleocytoplasmic transporter for viral mRNAs in which the E4orf6 protein directs both nuclear import and export. We now demonstrate that the E1B-55K protein itself shuttles efficiently in the absence of E4orf6. In addition, E1B-55K trafficking was independent of the defined shuttle proteins Mdm2 or p53, which interacts with E1B-55K. The identified N-terminal E1B-55K leucine-rich nuclear-export signal (NES) conferred rapid nuclear export even in a heterologous system in contrast to the postulated E4orf6NES. Interestingly, although shuttling was blocked by inhibitors of the CRM1 mediated export pathway, E1B-55K inhibited neither the activity nor the trafficking of the retroviral shuttle proteins HIV-1 Rev and HTLV-1 Rex. In contrast, Rev or Rex blocked the nuclear export of E1B-55K, most likely by competing for essential export factors. Our results provide new insights into the regulation of the adenovirus mRNA export system and the processes of adenovirus mediated transformation. Oncogene (2000) 19, 850 - 857.

[Transitory left ventricular outflow tract obstruction after mitral valve reconstruction]. / Passagere Obstruktion des linksventrikulären Ausflusstraktes nach Mitralklappenrekonstruktion.

HISTORY AND ADMISSION FINDINGS: A few days after uneventful surgical reconstruction of the mitral valve a 43-year-old man was found to have a systolic murmur due to prolapse of the posterior leaflet, suggesting renewed mitral regurgitation. INVESTIGATIONS: Echocardiography revealed haemodynamically significant left ventricular outflow tract obstruction (LVOT) with a left ventricle to aorta systolic gradient of 83 mm Hg. In addition there was moderately severe mitral regurgitation as well as a pericardial effusion but no signs of tamponade. TREATMENT AND COURSE: The obstruction was at first treated with verapamil, later with sotalol. The pericardial effusion was interpreted as part of a postcardiotomy syndrome. The effusion regressed under steroid administration, and the LVOT and mitral regurgitation also decreased. A provocation test five months postoperatively no longer brought about an outflow gradient. The good results were still present 12 months postoperatively. CONCLUSION: The described, rarely seen form of LVOT was probably caused by a combination of a very large anterior mitral leaflet, postoperative pericardial effusion and pharmacological effects. If the obstruction first occurs postoperatively, appropriate medication may improve the cardiac status and reoperation may be avoided. Echocardiography is an important method of diagnosis and serial monitoring.

Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 rex mutants.

The HTLV-1 Rex protein is an essential shuttle protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans-dominant (TD) mutant Rex proteins have been reported, however, the mechanism of trans-dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green fluorescent protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells. The presence of Rexp21-GFP inhibited the nuclear export of Rex and HIV-1 Rev as assayed by cotransfection and microinjection experiments. However, Rex-GFP or Rexp21-GFP did not form heteromultimers with nuclear Rex mutants in vivo. In contrast, shuttling was essential for trans-dominance. Thus, we propose that TD Rex mutants do not function by retaining WT Rex in the nucleus by protein-protein interactions, as demonstrated for Rev, but to titrate factors essential for Rex/Rev export. Our findings demonstrate differences between the regulatory proteins Rex and Rev and implicate a novel strategy to generate highly TD Rex mutants also applicable to other proteins.

Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells.

We established a straightforward experimental system to investigate directly the requirements for nucleocytoplasmic transport in live cells. For this purpose, substrates were created containing nuclear localization signals (NLS) or nuclear export signals (NES) linked to a chimeric protein composed of the glutathione S-transferase (GST) fused to the green fluorescent protein (GFP). The combination of GST/GFP-tagging allowed us to control protein expression in bacteria and to monitor protein purification during chromatography. Following microinjection into somatic cells, nuclear export/import of the highly fluorescent substrates could be observed directly by fluorescence microscopy. This system sets the stage to quantitate, in real time, the kinetics of nuclear import/export in living cells and to evaluate qualitative differences in various NLS/NES signals and pathways.

Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.

Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals.

Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

Multimer formation is not essential for nuclear export of human T-cell leukemia virus type 1 Rex trans-activator protein.

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.

Functional analysis of the human immunodeficiency virus type 1 Rev protein oligomerization interface.

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.

Interaction of the HIV-1 rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5.

It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.

Eukaryotic initiation factor 5A activity and HIV-1 Rev function.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine-modified protein stimulates synthesis of methionyl-puromycin in an in vitro assay which mimics the formation of the first peptide bond during protein synthesis, although the exact role of eIF-5A in vivo is still unknown. The unexpected finding that eIF-5A is a cellular cofactor of the HIV-1 Rev trans-activator protein may, however, provide a novel opportunity to reveal precisely what function eIF-5A performs in eukaryotic cells. In this review article, we first present a brief description of HIV-1 Rev function, followed by an overview of the data that identified eIF-5A as a Rev cofactor and, finally, discuss novel findings with respect to cellular eIF-5A activities.