Olaf Rosorius

Mature Dendritic Cells Infected with Herpes Simplex Virus Type 1 Exhibit Inhibited T-Cell Stimulatory Capacity

ABSTRACT 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 theHerpesviridae 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.

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.

Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation.

ABSTRACT Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

A novel transferable nuclear export signal mediates CRM1‐independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69

The best studied nuclear export processes are mediated by classical leucine‐rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the β‐herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus‐encoded cofactors. In contrast to proteins containing a leucine‐rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B‐insensitive export signal that is distinct from other export signals described previously and is required for pUL69‐mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.

Nuclear localisation of the G-actin sequestering peptide thymosin β4

Thymosin β4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin β4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin β4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin β4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

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.

Hemofiltrate CC Chemokine 1[9-74] Causes Effective Internalization of CCR5 and Is a Potent Inhibitor of R5-Tropic Human Immunodeficiency Virus Type 1 Strains in Primary T Cells and Macrophages

ABSTRACT Proteolytic processing of the abundant plasmatic human CC chemokine 1 (HCC-1) generates a truncated form, HCC-1[9-74], which is a potent agonist of CCR1, CCR3, and CCR5; promotes calcium influx and chemotaxis of T lymphoblasts, monocytes, and eosinophils; and inhibits infection by CCR5-tropic human immunodeficiency virus type 1 (HIV-1) isolates. In the present study we demonstrate that HCC-1[9-74] interacts with the second external loop of CCR5 and inhibits replication of CCR5-tropic HIV-1 strains in both primary T cells and monocyte-derived macrophages. Low concentrations of the chemokine, however, frequently enhanced the replication of CCR5-tropic HIV-1 isolates but not the replication of X4-tropic HIV-1 isolates. Only HCC-1[9-74] and HCC-1[10-74], but not other HCC-1 length variants, displayed potent anti-HIV-1 activities. Fluorescence-activated cell sorter analysis revealed that HCC-1[9-74] caused up to 75% down-regulation of CCR5 cell surface expression, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) achieved a reduction of only about 40%. Studies performed with green fluorescent protein-tagged CCR5 confirmed that both HCC-1[9-74] and RANTES, but not full-length HCC-1, mediated specific internalization of the CCR5 HIV-1 entry cofactor. Our results demonstrate that the interaction with HCC-1[9-74] causes effective intracellular sequestration of CCR5, but they also indicate that the effect of HCC-1[9-74] on viral replication is subject to marked cell donor- and HIV-1 isolate-dependent variations.

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. J. Cell. Biochem. 80:388–396, 2001.

Interaction of eukaryotic initiation factor 5A (eIF-5A) with nuclear pore complexes

The HIV-I Rev tram-activator protein is essential for the nuclear export of viral mRNAs. In the nucleus, Rev binds to the Rev Response Element (RRE) RNA target sequence, multimerizes and interacts with cellular cofactor(s), including the nucleoporin-like protein hRlP/Rab, CRMI and elF-SA. In particular, mutants of eiF-5A block nuclear export of Rev and thereby virus replication in human cells. Furthermore, immunological studies have demonstrated that eIF-SA is present both in the cytoplasm and the nucleus of mammalian ceils. In view of the proposed involvement of eIF-5A in RNA export processes, it was reasonable to assume that this factor inrcracts directly with nuclear pore complexes. We have studied the distribution of eIF-SA by a combination of immunofluorescence microscopy, immunogold EM and microinjection experiments using cultured mammalian cells (Vera) and Xenopus oocytes. in Vero cells, double-latreling experiments using antibodies reacting with nuc!eoporins (e.g. ~62) and eiF-SA demonstrated colocalizUion of eIF-SA with nuclear pore complexes. By immunogold EM, eIF-SA was found to be enriched at the nucteoplasmic face of the nuclear Pore complexes. In a second series of experiments, we demonstrated biochemically the presence of elF-5A in amphibian oocytes by Western analysis. Moreover, anti-elF-5A antibodies brightly stained the nuclear envelope in cryosections of Xenopus oocytes. At the EM level, eIF-SA was specifically enriched at the filaments extending from the inner ring of the Pore complexes into the nucleoplasm. To identify Potential interaction partners of elF-SA, we used a nitmcellulose overlay assay with nuclear envelope proteins of Xenopus oocytes. Several reacting bands were detected, including the nucleoporin ~62. Finally we provide direct evidence for an active nucleo/cytoplasmic transpoit of eIF5A: When we expressed (-Gal/eIF-5A fusion proteins in Vero cells. the protein became specifically enriched in the nucleus. Furthermore, micminiectiti of GST-eIF-SA fusion orotein into Vero cell nuclei resulted in the &pid translocation of the protein to the cytoplasm. This export was inhibited in the presence of CiTP(S, by tow temperature and the presence of Leptomycin B (an inhibitor of Rev export thaf binds CRMI). In sum, our data show that eIF-SA is able to actively enter and leave the cell nucleus. Moreover, eIF-SA interacts directly with components of rhe nuclear Pore complex. Thus, eIF-SA has characteristics that are typical for nuclear exmn factors.