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. 2018 Sep 12;92(19):e00842-18.
doi: 10.1128/JVI.00842-18. Print 2018 Oct 1.

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1β by Preventing NLRP3 Inflammasome Assembly

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Free PMC article

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1β by Preventing NLRP3 Inflammasome Assembly

Takayuki Komatsu et al. J Virol. .
Free PMC article

Abstract

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1β (IL-1β) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1β maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1β than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1β secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1β activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1β secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1β activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.

Keywords: NLRP3; V protein; inflammasome; paramyxovirus.

Figures

FIG 1
FIG 1
Role of the NLRP3 inflammasome in the regulation of SeV V(−)-induced IL-1β secretion. (A to D and H) PMA-differentiated THP1 (THP1/PMA) cells in a 96-well plate, 24-well plate, or cell chamber were infected with SeV wt or SeV V(−) at a multiplicity of infection (MOI) of 3 for 24 h. (E and F) THP1/PMA cells in a 96-well plate were mock treated or treated with Casp-1 inhibitor VX-765 for 1 h and then infected with SeV wt or SeV V(−) at an MOI of 3 for 24 h. (G) THP1/PMA or THP1-defNLRP3/PMA cells in a 96-well plate were infected with SeV V(−) at an MOI of 3 for 24 h. mRNAs for pro-IL-1β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by real-time PCR (A). Pro-IL-1β levels in cell lysates (B and F) or IL-1β levels in cell supernatants (C, E, and G) were determined by ELISA. Mature IL-1β in supernatants or pro-IL-1β in lysates was determined by immunoblot (IB) analysis. (H) Subcellular localizations of NLRP3 and ASC were examined under confocal microscopy. The scale bars represent 10 μm. Data were derived from at least three or four independent experiments and are presented as mean values. SD are shown as error bars. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 2
FIG 2
Role of SeV V protein in inhibition of the NLRP3 inflammasome. (A) HEK293T cells were transfected with plasmids encoding NLRP3 inflammasome signaling molecules together with the indicated plasmid. At 24 h posttransfection, IL-1β levels in cell supernatants were determined by ELISA, whereas cells were immunoblotted with anti-Flag antibody to measure the expression of NLRP3, pro-Casp-1, pro-IL-1β, and ASC. (B) THP1/vector/PMA and THP1/VSeV/PMA cells were stimulated with LPS (5 ng/ml) plus ATP (0 or 5 mM) or with poly(dA·dT) (0 or 400 ng/ml). IL-1β levels in cell supernatants were determined 24 h after treatment (B). Data were derived from at least three or four independent experiments and are presented as mean values. SD are shown as error bars. ***, P < 0.001.
FIG 3
FIG 3
Interaction of SeV V protein with NLRP3 protein. (A) HEK293T cells were transfected with various combinations of the indicated plasmids. These cells were then lysed 24 h after transfection and the lysates were subjected to immunoprecipitation (IP) with anti-V5 or Flag antibody, followed by IB analysis with anti-Flag or V5 antibody. The whole-cell lysates prepared for IP were also subjected to IB with anti-Flag or anti-V5 antibody. (B) The THP1/VSeV/PMA cells were lysed, and the lysates were subjected to IP with anti-Flag, followed by IB analysis with anti-NLRP3 or anti-Flag antibody. Data are representative of those from three independent experiments.
FIG 4
FIG 4
Requirement of the NLRP3-binding ability of V protein for inhibition of the NLRP3 inflammasome. (A) Schematic diagram of the SeV V mutants Vn (aa 1 to 317), Vc (aa 318 to 384), and Vcys (C362S and C365R). (B) HEK293T cells were transfected with plasmids encoding NLRP3 inflammasome signaling molecules together with the indicated plasmid. Immunoprecipitation and immunoblot analysis were performed as described for Fig. 3A. Data are representative of those from three independent experiments. (C) HEK293T cells were transfected with plasmids encoding NLRP3 inflammasome signaling molecules together with the indicated plasmid. IL-1β levels in cell supernatants were determined as described for Fig. 2A. Data were derived from at least three or four independent experiments and are presented as mean values. SD are shown as error bars. ***, P < 0.001.
FIG 5
FIG 5
Effect of SeV V protein on NLRP3 inflammasome assembly. (A and B) HEK293T cells in an 8-well microscope glass slide with a removable 8-well chamber were transfected with plasmids encoding NLRP3-GFP and ASC-Flag, together with the indicated plasmid encoding VSeV-V5His. (A) Subcellular localizations of NLRP3-GFP (green), ASC-Flag (red), VSeV-VH (blue-green), and nuclei (DAPI; blue) were visualized by confocal microscopy. The scale bars represent 10 μm. (B) ASC speck-forming cells and VSeV-positive cells were quantified from five randomly selected fields, and the percentages of ASC speck- and VSeV-positive cells in total VSeV-positive cells are shown. Data were derived from at least three or four independent experiments and are represented as mean values. SD are shown as error bars. **, P < 0.01. (C) Experimental data on BRET signals at 24 h posttransfection were derived from three independent experiments and are represented as mean values. SD are shown as error bars. **, P < 0.01 versus transfection with empty vector.
FIG 6
FIG 6
Role of SeV V protein in NLRP3 self-oligomerization. (A and B) HEK293T cells in an 8-well microscope glass slide with a removable 8-well chamber were transfected with plasmids encoding NLRP3-GFP, together with the indicated plasmid encoding VSeV-Flag. (A) Subcellular localizations of NLRP3-GFP (green), VSeV-Flag (red), and nuclei (DAPI; blue) were visualized by confocal microscopy. The scale bars represent 10 μm. (B) NLRP3 speck-forming cells and VSeV-positive cells were quantified from five randomly selected fields, and the percentages of NLRP3 speck- and VSeV-positive cells in the total VSeV-positive cells are shown. Data were derived from at least three or four independent experiments and are presented as mean values. SD are shown as error bars. **, P < 0.01. (C) Experimental data on BRET signals at 24 h posttransfection were derived from three independent experiments and are presented as mean values. SD are shown as error bars. **, P < 0.01 versus transfection with empty vector.
FIG 7
FIG 7
Inhibition of NLRP3 inflammasome activation is shared by paramyxovirus V proteins. HEK293T cells were transfected with various combinations of the indicated plasmids. (A) The concentration of IL-1β in the culture supernatants was determined as described for Fig. 1C. (B) IP and IB were performed as described for Fig. 3A. (C) The effect of V protein of HPIV2 or NiV on ASC speck formation (left) or NLRP3 speck formation (right) was evaluated as described for Fig. 5B and 6B. (D) The effect of V protein of HPIV2 and NiV on intermolecular interactions between NLRP3 and ASC (right) or NLRP3 and NLRP3 (left) was evaluated as described for Fig. 5C and 6C. SD are shown as error bars. **, P < 0.01, and ***, P < 0.001, versus transfection with empty vector.

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