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Retroviral retention activates a Syk-dependent HemITAM in human tetherin.

ABSTRACT: Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosine kinase (Syk), which is required for downstream NF-κB activation, indicating that the tetherin cytoplasmic tail resembles the hemi-immunoreceptor tyrosine-based activation motifs (hemITAMs) found in C-type lectin pattern recognition receptors. Retroviral-induced tetherin signaling is coupled to the cortical actin cytoskeleton via the Rac-GAP-containing protein RICH2 (ARHGAP44), and a naturally occurring tetherin polymorphism with reduced RICH2 binding exhibits decreased phosphorylation and NF-κB activation. Thus, upon virion retention, this linkage to the actin cytoskeleton likely triggers tetherin phosphorylation and subsequent signal transduction to induce an antiviral state.

SUBMITTER: Galao RP

PROVIDER: S-EPMC4161388 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Retroviral retention activates a Syk-dependent HemITAM in human tetherin.

Galão Rui Pedro RP Pickering Suzanne S Curnock Rachel R Neil Stuart J D SJ

Cell host & microbe 20140901 3

Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosin ...[more]

PMID: 25211072

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Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.

Dataset Information

Retroviral retention activates a Syk-dependent HemITAM in human tetherin.

Publications

Retroviral retention activates a Syk-dependent HemITAM in human tetherin.

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