Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS 1‐caspase‐1 axis
2018; Springer Nature; Volume: 37; Issue: 18 Linguagem: Inglês
10.15252/embj.201899347
ISSN1460-2075
AutoresYanyan Zheng, Qingxiang Liu, Yaoxing Wu, Ling Ma, Zhenzhen Zhang, Tao Liu, Shouheng Jin, Yuanchu She, Yi‐Ping Li, Jun Cui,
Tópico(s)Malaria Research and Control
ResumoArticle31 July 2018free access Source DataTransparent process Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis Yanyan Zheng MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yaoxing Wu orcid.org/0000-0002-3639-0202 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ling Ma Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhenzhen Zhang Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Tao Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Shouheng Jin MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanchu She MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yi-Ping Li Corresponding Author [email protected] orcid.org/0000-0001-6011-3101 Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yanyan Zheng MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yaoxing Wu orcid.org/0000-0002-3639-0202 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ling Ma Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhenzhen Zhang Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Tao Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Shouheng Jin MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanchu She MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yi-Ping Li Corresponding Author [email protected] orcid.org/0000-0001-6011-3101 Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Author Information Yanyan Zheng1,‡, Qingxiang Liu1,‡, Yaoxing Wu1,‡, Ling Ma2, Zhenzhen Zhang2, Tao Liu1, Shouheng Jin1, Yuanchu She1, Yi-Ping Li *,2,3 and Jun Cui *,1 1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 2Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 3Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 20 87335085; E-mail: [email protected] *Corresponding author. Tel: +86 20 39943429; E-mail: [email protected] EMBO J (2018)37:e99347https://doi.org/10.15252/embj.201899347 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non-structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1 at Lys134, thus inhibiting the proteasomal degradation of caspase-1. The enhanced stabilization of caspase-1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future. Synopsis Zika virus promotes NLRP3 inflammasome activation by stabilizing caspase-1 to suppress cGAS-mediated type I IFN signaling. The non-structural protein NS1 enhances ZIKV-induced NLRP3 inflammasome activation. NS1 stabilizes caspase-1 by blocking its proteasomal degradation. NS1 recruits USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1 at Lys134. ZIKV enhances inflammasome activation to benefit its infection by inhibiting type I IFN signaling. NS1-mediated stabilization of caspase-1 promotes the cleavage of cGAS. Introduction Zika virus (ZIKV) is an arthropod-borne flavivirus in the Flaviviridae family, which was initially discovered from Rhesus macaque in Uganda in 1947 (Dick et al, 1952). ZIKV contains a positive-sense single-stranded RNA genome and is closely related to several other important viruses that cause disease globally, including Dengue (DENV), hepatitis C, yellow fever, West Nile, and Japanese encephalitis viruses (Pierson & Diamond, 2013). ZIKV genome encodes a single polyprotein which can be processed to produce three structural (C, prM, and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (Pierson & Diamond, 2013). ZIKV infection was originally thought as a mild and self-limiting viral illness and caught little attention (Rossi et al, 2016; Miner & Diamond, 2017). However, it became a global health emergency since accumulating evidence has suggested that ZIKV infection is associated with the increasing incidence of microcephaly in newborns and Guillain–Barré syndrome during the outbreak of ZIKV in Brazil from 2015 (Ioos et al, 2014; Petersen et al, 2016; Rubin et al, 2016). Type I interferon (IFN) response serves as the first line of defense to combat viral infection (Schneider et al, 2014). Recently, we and other groups have demonstrated that ZIKV evolved several strategies to counter human IFN antiviral response (Fernandez-Garcia et al, 2009; Grant et al, 2016; Kumar et al, 2016; Wu et al, 2017; Xia et al, 2018). The IFN-antagonistic strategies can be mainly divided into two types (Fernandez-Garcia et al, 2009): The first strategy employed by ZIKV is to reduce and delay the activation of IFN production. For example, we recently reported that NS1 and NS4B of ZIKV blocked virus-mediated IFN signaling by targeting TBK1 (Wu et al, 2017). Xia et al showed the similar result of NS1 and found that residue 188 is critical for the inhibition of IFN (Xia et al, 2018). ZIKV could also utilize the other strategy to evade innate immunity by antagonizing IFN-mediated downstream signaling transduction. It has been reported that NS5 of ZIKV promoted the degradation of STAT2 (Grant et al, 2016; Kumar et al, 2016), while our study also revealed that NS2B3 can degrade JAK1 (Wu et al, 2017), thus inhibiting JAK-STAT signaling and coincidentally impairing downstream ISG expressions. Taken together, different non-structural proteins of ZIKV attenuate innate antiviral response at different levels of IFN signaling pathway and cooperatively assist ZIKV to evade host immune response (Bowen et al, 2018). To infect the fetus and affect the neural development of fetus, ZIKV should first cross the placental barrier and reach the fetus (Li et al, 2016; Miner et al, 2016). However, there is no commonly accepted mechanism employed by ZIKV to achieve mother-to-fetus transmission so far. One of the possible mechanisms utilized by ZIKV is to use monocytes as the carrier (Parekh et al, 2010; Khaiboullina et al, 2017). Monocytes are able to detect several kinds of pathogens and respond with the activation of inflammasome, a large signaling protein complex whose assembly usually requires the protein apoptosis-associated speck-like protein containing a CARD (ASC), caspases, and scaffold proteins (such as NLRP3 or AIM2) (Martinon et al, 2009). Once activated, the inflammasome triggers the activation of the cysteine protease caspase-1 to prompt the maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Park et al, 2007; Liu et al, 2016). IL-1β serves the central role in inflammatory response and initiates a series of innate immune responses (Dinarello, 2009). Recently, accumulating evidence has revealed that ZIKV can infect monocytes and result in the activation of inflammasome pathway (Khaiboullina et al, 2017; Tricarico et al, 2017; Wang et al, 2018). However, the correlation between inflammasome activation and ZIKV infection remains incompletely understood. Here, we report that ZIKV activates host inflammasome responses by increasing the stabilization of caspase-1. Interestingly, ZIKV NS1 targets caspase-1 and removes its K11-linked ubiquitin chains at lysine (Lys) 134 by recruiting deubiquitinase (DUB) USP8. Consequently, caspase-1 targets to cGAS for cleavage, which results in reduced type I IFN signaling and enhanced ZIKV replication. Furthermore, NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKV in vivo. Taken together, we identify a novel function of ZIKV NS1 in regulating the stability of caspase-1 and therefore reveal a mechanism by which ZIKV evades host antiviral response via initiating inflammasome activation. Our findings will facilitate the development of antiviral inhibitors and vaccine design toward novel strategies against ZIKV infection. Results ZIKV infection induces NLRP3 inflammasome activation The inflammasome plays a key role in host innate immune responses by promoting pro-caspase-1 cleavage to generate the active subunits p20 and p10, leading to the maturation and secretion of IL-1β. In order to determine whether ZIKV infection activates the inflammasomes, we measured IL-1β secretion from unprimed or lipopolysaccharide (LPS)-primed THP-1 cells (a human monocyte cell line) infected with ZIKV (Asian lineage strain GZ01 (GenBank No. KU820898) if not specified). Immunoblot and ELISA analyses demonstrated that ZIKV infection induced the release of IL-1β from both unprimed and LPS-primed THP-1 cells (Fig 1A and B). NLRP3 and AIM2 are known to play important roles in inflammasome activation by virus infection (Martinon et al, 2009). We next generated NLRP3 and AIM2 knockout (KO) THP-1 cells by CRISPR/Cas9 technology (Fig EV1A), and KO efficiency was functionally validated by LPS plus ATP or poly (dA:dT) treatment as positive or negative controls. We found that ZIKV-induced IL-1β and IL-18 secretion as well as caspase-1 cleavage was abrogated in NLRP3 KO THP-1 cells but not in AIM2 KO THP-1 cells (Figs 1C and EV1B–D). To further confirm the induction of NLRP3 inflammasome by ZIKV, we examined IL-1β secretion in two different clones of NLRP3 or AIM2 KO cells at various time points after ZIKV infection and got consistent results (Figs 1D and EV1E). Moreover, we found that knockdown of NLRP3 by two different siRNAs suppressed IL-1β secretion after ZIKV infection in human peripheral blood mononuclear cells (PBMCs) from two healthy donors (Figs 1E and EV1F). We also examined another ZIKV recombinant (MR766)—a historical African lineage strain (Dick et al, 1952; Schwarz et al, 2016), which differs from GZ01 by 11% at nucleotide level. We found that both African strain MR766 and Asian strain GZ01 induced NLRP3 inflammasome activation (Fig 1F). In addition, we compared ZIKV with Dengue virus serotype 2 (DENV-2) strain 16681 (Pu et al, 2011), a flavivirus closely related to ZIKV, and found that compared with ZIKV, DENV-2 only induced weak inflammasome activation (Fig 1F). Taken together, these results demonstrate that ZIKV infection specifically activates the NLRP3 inflammasome. Figure 1. The non-structural protein NS1 of ZIKV enhances ZIKV-induced NLRP3 inflammasome activation ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 h) THP-1 cells infected with ZIKV (MOI = 1) for 36 h. Uninfected cells serve as mock control. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated, or primed by LPS (500 ng/ml, 3 h), after ZIKV infection (MOI = 1) for 36 h. ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1, or AIM2 KO#1 THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI = 1) at the indicated time points. ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI = 1) for 36 h. ELISA of IL-1β in the supernatant of WT, NLRP3 KO, or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI = 1) or DENV-2 (MOI = 1) infection for 36 h. ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 h or mock control. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) treatment for 6 h. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI = 1) or mock control for 24 h. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) stimulation for 6 h. Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1, and pro-IL-1β together with NS1. Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 h. ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h) stimulation. Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h) stimulation. Data information: In (B, H–J, L), data are representative of three independent experiments. In (A, C, D–G, K), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj201899347-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The non-structural protein NS1 of ZIKV enhances ZIKV-induced NLRP3 inflammasome activation, related to Fig 1 A. Immunoblot analysis of extracts of wild-type (WT) and two different clones of NLRP3 or AIM2 knockout (KO) THP-1 cells by the indicated antibodies. B. ELISA of IL-18 in the supernatants of WT, NLRP3 KO#1, or AIM2 KO#1 THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). C, D. Immunoblot analysis of the cleavage of caspase-1 and IL-1β in WT, NLRP3 KO#1 (C) or AIM2 KO#1 (D) THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). E. ELISA of IL-1β in the supernatants of two clones of AIM2 KO THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. F. Immunoblot analysis of extracts of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI = 1) for 36 h by the indicated antibodies. G. THP-1 cells were pre-infected with ZIKV (MOI = 1) for 12 h. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) stimulation for the indicated time points. Cell death was measured by LDH (lactate dehydrogenase) release. H. Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids encoding NLRP3, ASC, pro-caspase-1, and pro-IL-1β together with NS2B3 (left) or NS4B (right). I. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of Flag-NS1-inducible unprimed THP-1 cells stimulated by LPS (500 ng/ml, 3 h), ATP (5 mM, 6 h), or poly (I:C) (2 mg/ml, 6 h), respectively. Data information: In (A, C, D, F, H, I), data are representative of three independent experiments. In (B, E, G), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; ***P < 0.001. Download figure Download PowerPoint A number of viruses have evolved mechanisms to prolong their intracellular survival by inhibiting the inflammasome activation (Komune et al, 2011; Cheong et al, 2015). However, we observed that ZIKV infection promotes the activation of inflammasomes, since more cleaved IL-1β was detected in THP-1 cells infected with ZIKV followed by LPS and ATP treatment to activate NLRP3 inflammasome (Fig 1G and H). Meanwhile, ZIKV infection did not affect the inflammasome activation-induced cell death (Fig EV1G). Together, these results suggest that ZIKV infection triggers NLRP3 inflammasome activation. The non-structural protein NS1 of ZIKV enhances inflammasome activation Next, we set out to unveil the underlying mechanisms through which ZIKV promotes inflammasome activation. The non-structural protein 1 (NS1) is the major host-interaction molecule that functions in flavivirus replication, pathogenesis, and immune evasion (Hilgenfeld, 2016). To evaluate whether NS1 of ZIKV plays a role in inflammasome activation, we utilized human embryonic kidney (HEK) 293T cells in which the NLRP3 inflammasome is deficient and can be reconstituted. Overexpression of NS1 but not NS2B3 or NS4B of ZIKV led to a profound inflammasome activation, as characterized by the increased secretion of mature caspase-1 and IL-1β into the culture medium (Figs 1I and EV1H). To further confirm these results, we generated a doxycycline (Dox)-inducible NS1 THP-1 cell line (Fig 1J). After LPS priming and ATP or poly (I:C) stimulation, more mature IL-1β and cleaved caspase-1 could be detected when NS1 was induced to express (Figs 1K and L, and EV1I). Taken together, these results demonstrate that NS1 further facilitates NLRP3 inflammasome activation during ZIKV infection. The enhanced inflammasome activation by NS1 benefits ZIKV infection In light of the critical role of the inflammasome in the host antiviral responses, we turned our attention to its impact on ZIKV replication. Surprisingly, we observed a significantly higher accumulation of ZIKV viral RNA in LPS and ATP pre-treated THP-1 cells (Fig 2A). Consistently, knockout (KO) of NLRP3 in THP-1 cells resulted in lower viral replication (Fig 2B). To further confirm this finding, we examined ZIKV replication in bone marrow-derived macrophages (BMDMs) from NLRP3-deficient (Nlrp3−/−) mice (Fig 2C) by qRT–PCR as well as plaque titration assay. The results showed that the level of ZIKV RNA (Fig 2D) and virus titer (Fig 2E) was significantly lower in Nlrp3−/− BMDMs, compared to that of wild-type (WT) BMDMs. We next generated caspase-1 KO THP-1 cells (Fig 2F) and found that caspase-1 deficiency also decreased ZIKV replication (Fig 2G). Moreover, the use of Ac-YVAD-cmk (YVAD for short), a specific inhibitor of caspase-1, attenuated ZIKV replication (Fig EV2A), indicating that the reinforcing effect of NLRP3 inflammasome on ZIKV replication relied on the enzyme activity of caspase-1. Consistently, NS1-induced up-regulation of ZIKV replication could be blocked by YVAD (Fig 2H), indicating that NS1 also functions through caspase-1 activity. To validate these findings in human primary cells, we knocked down NLRP3 by siRNA in PBMCs from two different healthy donors (Fig 2I). We observed lower viral replication after knockdown of NLRP3 (Fig 2J). Furthermore, YVAD treatment in PBMCs also suppressed ZIKV replication (Fig 2K). We next examined whether MR766 strain shared the same property as GZ01 strain and found that NLRP3 deficiency inhibited the viral replication of both strains (Fig EV2B). Since NLRP3 inflammasome activation is sufficient to drive pro-IL-1β processing and secretion, we therefore assessed the contribution of IL-1β to this course. However, we found that the replication of ZIKV was not affected by IL-1β treatment (Fig EV2C). Taken together, these data suggest that the enhanced NLRP3 inflammasome activation by NS1 benefits to ZIKV replication, which is dependent on the activity of caspase-1 but not IL-1β. Figure 2. NS1 enhances inflammasome activation to benefit ZIKV infection Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 h) and ATP (5 mM, 6 h), followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. Immunoblot analysis of extracts of WT or Nlrp3−/− BMDMs by the indicated antibodies. Relative qRT–PCR analysis of ZIKV RNA in WT or Nlrp3−/− BMDMs infected with ZIKV (MOI = 1) for the indicated time points. Plaque titration of ZIKV in supernatants of WT or Nlrp3−/− BMDMs infected with ZIKV (MOI = 1) for the indicated time points. Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies. Relative qRT–PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μM) for 3 h followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors. Relative qRT–PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI = 1) for 36 h. qRT–PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20 μM) for 3 h followed by ZIKV infection (MOI = 1) for 36 h. Data information: In (C, F), data are representative of three independent experiments. In (A, B, D, E, G–K), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. NS1 enhances inflammasome activation by inhibiting the proteasomal degradation of caspase-1 to benefit ZIKV infection, related to Figs 2 and 3 Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with Ac-YVAD-cmk (20 μM) for 3 h, followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (GZ01) or ZIKV (MR766) (MOI = 1) for 36 h. Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with IL-1β (2.5 ng/ml) for 3 h, followed by ZIKV infection (MOI = 1) for the indicated time points. Immunoblot analysis of THP-1 cells infected with ZIKV (GZ01), ZIKV (MR766), or DENV-2 (MOI = 1) for 36 h by the indicated antibodies. Intensity analysis of the bands in Fig 3F from the three independent experiments. Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), 3-methyladenine (3MA) (10 mM), chloroquine (CQ) (50 mM), or NH4Cl (20 mM) for 6 h. Confocal microscopy analysis of the co-localization of proteasome subunit PSMD14 and caspase-1 in HeLa cells transfected with or without NS1 followed by CHX (100 μg/ml) and MG132 (10 mM) treatment for 3 h. Scale bar is 10 μm. Intensity analysis of the bands in Fig 3I from three independent experiments. Data information: In (D, F), data are representative of three independent experiments. In (A–C, E, H), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; ***P < 0.001. Download figure Download PowerPoint NS1 inhibits the proteasomal degradation of caspase-1 To dissect the molecular mechanisms by which NS1 enhances inflammasome and caspase-1 activation, we sought to determine whether NS1 directly interacts with inflammasome components. Co-immunoprecipitation experiments showed that NS1 had a strong interaction with caspase-1 in 293T (Fig 3A) and Flag-NS1-inducible THP-1 cells (Fig 3B). Meanwhile, we repeatedly observed an increased protein level of caspase-1 after increasing the effect of NS1 on caspase-1 abundance, and we repeated this work both in THP-1 and in 293T cells. After NS1 overexpression, we observed a considerable accumulation of caspase-1 but not of any other proteins such as IL-1β and NLRP3 (Fig 3C and D). In addition, RT–PCR showed that caspase-1 mRNA abundance was not altered with NS1 overexpression (Fig 3D). We did not observe the accumulation effect of caspase-1 by DENV-2 NS1 protein, although DENV is a flavivirus closely related to ZIKV (Fig 3E). In line with this result, we observed increased protein abundance of caspase-1 after the infection of both strains of ZIKV but not DENV-2 (Fig EV2D). We next performed cycloheximide (CHX) chase assay to determine whether NS1 affects the stability of caspase-1 (Figs 3F and EV2E) and found that NS1 stabilized caspase-1 protein by delaying its degradation. To reveal which degradation system is responsible for the degradation of caspase-1, we assessed the caspase-1 stability in the presence of different inhibitors and found that NS1-mediated stabilization of caspase-1 was blocked by the proteasome inhibitors MG132, lactacystin, or carfilzomib, but not by the autophagy inhibitor (3MA) or lysosome inhibitor chloroquine (CQ) and NH4Cl (Figs 3G and EV2F). We also performed confocal microscopy analysis to visualize the co-localization of caspase-1 with the proteasome and observed that caspase-1 co-localized with the proteasome subunit PSMD14. However, the co-localizations were diminished in the presence of NS1, suggesting that the degradation of caspase-1 via proteasome pathway can be inhibited by NS1 (Fig EV2G). Collectively, these results demonstrate that NS1 protects caspase-1 from proteasomal degradation. Figure 3. NS1 inhibits the proteasomal degradation of caspase-1 Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1, and Flag-IL-1β. WCL, whole-cell lysates. Co-immunopre
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