6 research outputs found
Model depicting the mechanisms used by CoV PLPs to block STING from signaling the activation of the IFN-β induction pathway.
<p>(<b>A</b>) Activation of sensors such as RIG-I induces interaction with the signaling complex including MAVS, STING, IRF-3 and TBK-1. Activated MAVS interacts with STING, which dimerizes, leading to the activation of IKK complex, TBK1 and IKKε <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030802#pone.0030802-Zhong1" target="_blank">[8]</a>. The activation of this complex leads to the ubiquitination of RIG-I, STING, IRF-3 and TBK1 and the phosphorylatin of STING and IRF-3. Activated the transcription factor IRF-3 translocates to the nucleus inducing production of IFN. (<b>B</b>) Coronavirus papain-like protease domains (depicted here as PLP) interact with STING to block signaling by blocking assembly or stability of STING dimers and preventing the ubiquitination of signaling proteins, such as RIG-I, TBK1, and IRF-3.</p
Coronavirus NL63 PLP2-TM associates with STING and nsp3 co-localizes with STING in virus-infected cells.
<p>(<b>A</b>) HEK293T cells were cotransfected with plasmid DNAs expressing STING-Flag and either wild type or catalytic mutants of NL63-PLP2-TM-V5. Cell lysates were prepared at 28 hrs post-transfection and subjected to immunoprecipitation (IP) with anti-Flag antibody. The products of the immunoprecipitation were separated by SDS-PAGE and subjected to immunoblotting (IB). STING-Flag, PLP2-TM-V5 and the catalytic mutant expression were selectively detected from whole cell lysates (WCL) using anti-Flag and anti-V5 antibodies. (<b>B</b>) HEK293-ACE2 cells were transfected with STING-V5 for 4 hours and then infected with HCoV-NL63 for 24 hrs and evaluated for expression of and localization of replicase product nsp3 (anti-nsp3, red) and STING-V5 (anti-V5, green).</p
NL63 PLP2-TM disrupts signaling complex formation.
<p>HEK293T cells were co-transfected with STING-HA together with either Flag-tagged RIG-I (<b>A</b>), Flag-tagged MAVS (<b>B</b>) or Flag-tagged IKKε (<b>C</b>), and PLP2-TM-V5. At 28 h after transfection, cell lysates were prepared and subjected to immunoprecipitate (IP) and immunoblot (IB) with the indicated antibodies. The asterisk indicates the nonspecific band.</p
Reduction of ubiquitinated forms of RIG-I, STING, TBK1 and IRF-3 in the presence of NL63 PLP2-TM.
<p>HEK293 cells were transfected with Flag-tagged RIG-I(<b>A</b>), TBK1(<b>B</b>), myc-IRF-3(<b>C</b>), or STING-Flag (<b>D</b>) together with plasmid DNA expressing HA-tagged Ub in the presence or absence of V5-tagged PLP2-TM-V5. Cells were incubated for 24 hours after transfection and treated with 25 µM MG132 for 4 hours prior to harvesting lysates. Lysates were immunoprecipitated with the indicated antibody and the products were subjected to immunoblotting with anti-HA to evaluate ubiquitinated proteins (upper panels). The whole cell lysates (WCL) were blotted to evaluate expression of each epitope-tagged product (bottom panels).</p
Expression of coronavirus PLPs blocks STING-mediated activation of the interferon stimulated response element (ISRE).
<p>(<b>A</b>) Schematic diagram of human coronaviruses (HCoV) NL63 illustrating the processing of replicase polyproteins to generate nonstructural proteins (nsp's). The papain-like protease domains, the catalytic residues that essential for protease catalytic activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030802#pone.0030802-Chen1" target="_blank">[35]</a>, and the transmembrane (TM) domain within nsp3 are indicated. (<b>B</b> and <b>C</b>) HEK293T cells were transfected with the STING-HA, ISRE-luc reporter and either wild-type or catalytic mutants of HCoV-NL63 PLP2-TM or SARS-CoV PLpro-TM. Asterisks indicate statistical significance (P<0.05) in comparison with ISRE-reporter activity stimulated with STING. (<b>D</b>) Immunofluorescence microscopy of HEK-293T cells expressing STING-HA and PLP2-TM-V5. Cells were fixed at 24 hrs post-transfection and the localization of endogenous IRF-3 (anti-IRF-3, green) and the epitope-tagged products was visualized by confocal microscopy.</p
NL63 PLP2-TM interacts with STING and disrupts STING dimers.
<p>(<b>A</b>) HEK293T cells were co-transfected with plasmid DNA expressing STING-Flag, and/or PLP2-TM and/or infected with Sendai virus (SeV) as indicated above. The cell lysates were separated by SDS-PAGE and subjected to immunoblotting with antibodies as indicated on the left. (<b>B</b>) HEK293-ACE2 cells were transfected with plasmid DNA expressing STING-HA and infected with SARS-CoV as indicated and cell lysates were subjected to immunoprecipitation with anti-HA. The immunoprecipitated products were analyzed by SDS-PAGE and immunoblotted to access STING monomers and dimers. Whole cell lysates were immunoblotted to detected SARS-CoV replicase protein nsp3 and STING-HA. (<b>C</b>) Cells were co-transfected with STING-HA and STING-Flag with either wild-type or the indicated catalytic mutant of PLP2-TM and lysates were immunoprecipitated (IP) and immunoblotted (IB) to detect expression of each product.</p