Differential Ligand Binding to a Human Cytomegalovirus Chemokine Receptor Determines Cell Type–Specific Motility

While most chemokine receptors fail to cross the chemokine class boundary with respect to the ligands that they bind, the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 binds multiple CC-chemokines and the CX3C-chemokine Fractalkine. US28 binding to CC-chemokines is both necessary and sufficient to induce vascular smooth muscle cell (SMC) migration in response to HCMV infection. However, the function of Fractalkine binding to US28 is unknown. In this report, we demonstrate that Fractalkine binding to US28 not only induces migration of macrophages but also acts to inhibit RANTES-mediated SMC migration. Similarly, RANTES inhibits Fractalkine-mediated US28 migration in macrophages. While US28 binding of both RANTES and Fractalkine activate FAK and ERK-1/2, RANTES signals through Gα12 and Fractalkine through Gαq. These findings represent the first example of differential chemotactic signaling via a multiple chemokine family binding receptor that results in migration of two different cell types. Additionally, the demonstration that US28-mediated chemotaxis is both ligand-specific and cell type–specific has important implications in the role of US28 in HCMV pathogenesis

PANDA Phase One - PANDA collaboration

The Facility for Antiproton and Ion Research (FAIR) in Darmstadt, Germany, provides unique possibilities for a new generation of hadron-, nuclear- and atomic physics experiments. The future antiProton ANnihilations at DArmstadt (PANDA or P¯ANDA) experiment at FAIR will offer a broad physics programme, covering different aspects of the strong interaction. Understanding the latter in the non-perturbative regime remains one of the greatest challenges in contemporary physics. The antiproton–nucleon interaction studied with PANDA provides crucial tests in this area. Furthermore, the high-intensity, low-energy domain of PANDA allows for searches for physics beyond the Standard Model, e.g. through high precision symmetry tests. This paper takes into account a staged approach for the detector setup and for the delivered luminosity from the accelerator. The available detector setup at the time of the delivery of the first antiproton beams in the HESR storage ring is referred to as the Phase One setup. The physics programme that is achievable during Phase One is outlined in this paper

Mouse Cytomegalovirus M33 Is Necessary and Sufficient in Virus-Induced Vascular Smooth Muscle Cell Migration

Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration

Fractalkine induces US28-mediated migration of macrophages.

<p>(A) Expression of US28 was determined via western blot analysis of total cellular lysate for the HA epitope tag. 2×10⁵ rat macrophages were infected for 72 hours with US28-HA adenovirus vector at the indicated MOI. (B) The efficiency of adenovirus transduction was determined by FACS analysis of permeablized rat macrophages infected for 72 hours at MOI 250 with US28-HA adenovirus vector. (C) Surface expression of US28 was confirmed via FACS analysis of non-permeablized rat macrophages infected for 72 hours at MOI 250 with US28-HA adenovirus vector. <i>In vitro</i> migration assays were performed on 1×10⁵ Ad-US28 and/or Ad-Trans infected rat macrophages treated with the indicated concentrations of (D) Fractalkine or (E) RANTES. For all conditions, n≥12 from four independent experiments. Percentages are calculated relative to unstimulated macrophages infected with adenovirus transactivator (Trans). (F) Competition migration assays were performed on Ad-US28 expressing macrophages treated with 40ng/ml of Fractalkine and the indicated concentrations of RANTES as a competing ligand. For all conditions, n≥12 from two independent experiments. Percentages are calculated relative to unstimulated macrophages infected with Ad-Trans.</p

RANTES and Fractalkine activation of FAK is dependent on different G-proteins.

<p>(A) FAK activity in FAK −/− cells expressing both US28 and wt-FAK in response to either Fractalkine or RANTES and in the presence or absence of pertussis toxin was assessed by Grb2/FAK co-immunoprecipitation reactions. Cells were harvested in modified RIPA buffer at 0 (unstimulated), 5, 10, 15, and 30 minutes post addition of ligand. Active FAK associated with Grb2 was visualized by western blotting for phospho-tyrosine. (B,C) The ability of Gα12 and Gαq to enhance or abrogate RANTES and Fractalkine mediated activation of FAK through US28 was assessed by overexpressing (B) Gα12 or (C) Gαq in FAK −/− cells. FAK −/− cells infected with adenovirus expressing US28, wt-FAK and Gα12 or Gαq were stimulated with either RANTES or Fractalkine. As in (A), FAK activity was assessed by Grb2/FAK co-immunoprecipitation reactions and active FAK associated with Grb2 was visualized by western blotting for phospho-tyrosine. Western blots were quantified by densitomitry and fold FAK activation compared to unstimulated control is indicated below each lane.</p

Fractalkine inhibits US28-mediated SMC migration induced by RANTES.

<p>(A) SMC migration assays were performed on cells infected with adenovirus expressing US28-HA treated with either RANTES or Fractalkine at the indicated concentrations. Data are represented as a percentage of unstimulated cells infected with control adenovirus transactivator only. For all conditions, n>6 from two independent experiments. (B) SMC migration assays were performed on US28-expressing cells treated with RANTES, Fractalkine or 40ng/ml of RANTES and the indicated concentrations of Fractalkine as a competing ligand.</p