Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity.

We present clinicopathologic data on 10 pulmonary myxoid sarcomas, which are defined by distinctive histomorphologic features and characterized by a recurrent fusion gene, that appear to represent a distinct tumor entity at this site. The patients [7 female, 3 male; aged 27 to 67 y (mean, 45 y)] presented with local or systemic symptoms (n=5), symptoms from cerebral metastasis (1), or incidentally (2). Follow-up of 6 patients showed that 1 with brain metastasis died shortly after primary tumor resection, 1 developed a renal metastasis but is alive and well, and 4 are disease free after 1 to 15 years. All tumors involved pulmonary parenchyma, with a predominant endobronchial component in 8 and ranged from 1.5 to 4 cm. Microscopically, they were lobulated and composed of cords of polygonal, spindle, or stellate cells within myxoid stroma, morphologically reminiscent of extraskeletal myxoid chondrosarcoma. Four cases showed no or minimal atypia, 6 showed focal pleomorphism, and 5 had necrosis. Mitotic indices varied, with most tumors not exceeding 5/10 high-power fields. Tumors were immunoreactive for only vimentin and weakly focal for epithelial membrane antigen. Of 9 tumors, 7 were shown to harbor a specific EWSR1-CREB1 fusion by reverse transcription-polymerase chain reaction and direct sequencing, with 7 of 10 showing EWSR1 rearrangement by fluorescence in situ hybridization. This gene fusion has been described previously in 2 histologically and behaviorally different sarcomas: clear cell sarcoma-like tumors of the gastrointestinal tract and angiomatoid fibrous histiocytomas; however, this is a novel finding in tumors with the morphology we describe and that occur in the pulmonary region.

Low frequency of the TEL/AML1 fusion gene in acute lymphoblastic leukaemia in Spain.

We report on a series of Spanish patients with acute lymphoblastic leukaemia in whom the t(12;21) [TEL/AML1] translocation could not be identified with two sensitive techniques: reverse transcript-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH). 101 cases were analysed: 38 children (29 B-cell precursor; nine T-cell precursor) and 63 adults (48 B-cell precursor; 15 T-cell precursor). Specific RT-PCR to amplify the TEL/AML1 fusion transcript was negative in all 101 cases. Moreover, all 38 paediatric samples were also negative by interphase FISH analysis for the presence of the TEL/AML1 fusion. These results suggest the existence of geographic/race variations in the genotype of acute lymphoblastic leukaemia (ALL).