Biomedical subjects

Alberto Fraile-Ramos

Publications and source records attributed to Alberto Fraile-Ramos.

4 recordsLinked to original sources

PubMed2006

Novel replication complex architecture in rubella replicon-transfected cells.

Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

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PubMed2003

Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway.

Arrestins bind phosphorylated G-protein coupled-receptors (GPCR) and inhibit agonist-induced signal transduction by uncoupling the receptors from their cognate G-proteins. beta-arrestins also act as adaptors that target GPCR to endocytic clathrin-coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of beta-arrestins in US28 trafficking, we used embryonic fibroblasts derived from beta-arrestin knockout mice. In these cells, the internalization of transfected beta2-adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28-mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin-mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the micro2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with micro2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody-feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin-mediated mechanism but is independent of beta-arrestins.

Animals↗

PubMed2002

Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes.

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.

Animals↗

PubMed2002

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

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