Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZ

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

Ecosystem-wide metagenomic binning enables prediction of ecological niches from genomes

The genome encodes the metabolic and functional capabilities of an organism and should be a major determinant of its ecological niche. Yet, it is unknown if the niche can be predicted directly from the genome. Here, we conduct metagenomic binning on 123 water samples spanning major environmental gradients of the Baltic Sea. The resulting 1961 metagenome-assembled genomes represent 352 species-level clusters that correspond to 1/3 of the metagenome sequences of the prokaryotic size-fraction. By using machine-learning, the placement of a genome cluster along various niche gradients (salinity level, depth, size-fraction) could be predicted based solely on its functional genes. The same approach predicted the genomes’ placement in a virtual niche-space that captures the highest variation in distribution patterns. The predictions generally outperformed those inferred from phylogenetic information. Our study demonstrates a strong link between genome and ecological niche and provides a conceptual framework for predictive ecology based on genomic data

rSgC and IgD enhance chemokine-dependent migration.

<p>(<b>A</b>) Transwell experiment showing the effect of rSgC or IgD proteins on CXCL12-α-induced migration. A range of chemokine concentrations alone or together with 1:200 molar ratio of chemokine:rSgC or IgD was incubated in the bottom chamber of the transwell during 30 minutes at 37°C in a humidified incubator prior to the addition of Jurkat T cells to the top chamber. Migrated cells were detected in the bottom chamber. Plots show one representative assay performed in triplicate out of at least three independent experiments. Error bars represent standard deviation. (<b>B</b>) Coomassie staining showing a representative purification of the IgD protein used in the chemotaxis experiments. ***<i>P<0</i>.<i>0005</i>.</p

VZV rSgC interacts with the cell surface through a specific interaction with GAGs.

<p>(<b>A</b>) Histograms showing the interaction of MHV-68 M3 (left panel), HSV-2 rSgG (middle panels) and VZV rSgC (right panels) with CHO-K1 cells (upper panels) or CHO-618 cells (lower panels). CHO-K1 cells contain GAGs whereas CHO-618 cells are devoid of GAGs. Surface-bound proteins were detected by flow cytometry using an anti His-tag antibody. Light grey histograms represent the signal obtained when no recombinant protein was used. Empty histograms represent the signal obtained with 100 ng of purified recombinant protein. (<b>B</b>) Graph showing the number of resonance units (R.U.) obtained when rSgC (alone or in the presence of increasing concentrations of heparin) was injected over an SA chip containing immobilised heparin. The maximum R.U., recorded at 90 seconds, is shown. The signal obtained with buffer alone was subtracted from the signal obtained with the samples containing rSgC. The ratios of rSgC:heparin used are indicated in the X axis. (<b>C</b>) Western blots showing binding of VZV rSgB (top blot), VZV rSgC (middle blot) or VZV rSgI (bottom blot) to heparin beads. Bound proteins were detected by Western blotting using an anti His-tag antibody. Binding was competed with increasing amounts of soluble heparin (0.1, 0.5, 1 and 2 mg). The input, corresponding to 1/10 of the starting material, is shown in the right lane. One representative experiment out of at least three independent experiments is shown in <b>A-C</b>. Abbreviations: Hep, heparin; Hep B, heparin beads; gp, glycoprotein; rSg, recombinant soluble glycoprotein; kDa, kiloDaltons.</p

Identification of the rSgC binding domain responsible for interaction with chemokines.

<p>(<b>A</b>) Schematic representation of full-length gC protein (top construct) and deletion constructs containing either amino acids 23–151 (R2D, middle construct) or amino acids 140–531 (IgD, bottom construct). The numbers indicate amino acid positions within VZV gC Dumas strain. To improve secretion in insect cells the VZV gC signal peptide (SP) was substituted by that of the honey bee melittin (HM). The introduction of the N-terminal histidine tag (His) allowed purification of the proteins by affinity chromatography. (<b>B</b>) Purified proteins were detected by Coomassie staining (upper panels) or by Western blotting (bottom panels) using antibodies: anti His-tag (left panel), anti R2D (middle panel) and anti IgD (right panel). Left and middle blots were obtained following transfer from the same gel, whereas the right blot comes from an independent gel. (<b>C,D</b>) Sensorgrams showing the association and dissociation phases of the interaction between chemokines (CXCL2, CXCL12-α, CXCL13, CCL19 and the negative control CX3CL1 at 100 nM) and IgD (<b>C</b>) or R2D (<b>D</b>). The same chemokines were injected in the IgD and R2D chips. The arrow indicates the end of the chemokine injection. (<b>D</b>) An antibody targeted to gC was injected into the R2D chip at a concentration of 10 ng/μl. Abbreviations: RU, resonance units; kDa, kiloDaltons; TMB, transmembrane; CD, cytoplasmic domain.</p

Characterization of the rSgC binding domain responsible for interaction with the cell surface.

<p>Histograms showing the interaction of purified recombinant M3, full-length rSgC, IgD and R2D with CHO-K1 cells (<b>A</b>) or CHO-618 cells (<b>B</b>). CHO-K1 cells contain GAGs whereas CHO-618 cells are devoid of GAGs. Bound proteins were detected by flow cytometry using an anti His-tag antibody. Light grey histograms represent the signal obtained when no recombinant protein was used. Empty histograms represent the signal obtained with recombinant protein. One representative experiment out of at least three independent experiments is shown.</p

VZV rSgC enhancement of chemokine activity requires interaction with the chemokine and subsequent signalling through the chemokine receptor.

<p>Transwell experiment showing the effect of pertussis toxin (PTX) (<b>A</b>) or AMD3100 (<b>B</b>) on the chemotaxis of Jurkat T cells towards increasing concentrations of CXCL12-α alone or in the presence of 1:200 molar ratio of chemokine:rSgC. The arrows in (<b>A, B</b>) point to the condition with rSgC only, without chemokine. Transwell experiment showing the migration of THP-1 cells (<b>C, D</b>) towards increasing concentrations of wild type or mutated CCL5 (<b>C</b>) or CCL3 and CCL5 (<b>D</b>) alone or in the presence of 1:200 molar ratio of chemokine:IgD (<b>C</b>) or chemokine:rSgC (<b>D</b>). In all experiments the chemokine was incubated alone or together with VZV rSgC at 37°C in a humidified incubator in the bottom chamber of the transwell prior to the addition of the leukocytes to the top chamber. Migrated cells were detected in the lower chamber at the end of the experiment. Plots show one representative assay performed in triplicate out of at least three independent experiments. Error bars represent standard deviation. *<i>P<0</i>.<i>05</i>; **<i>P<0</i>.<i>005</i>; ***<i>P<0</i>.<i>0005</i>.</p