Zika virus NS5 protein inhibits type I interferon signaling via CRL3 E3 ubiquitin ligase-mediated degradation of STAT2

Significance

The ZIKA virus (ZIKV) subverts the innate immune response by degrading STAT2 via NS5 protein, crucial for evading antiviral defenses. However, the precise mechanism has remained elusive. Here, we elucidated ZSWIM8 as the substrate receptor of the Cullin3-RING E3 ligase complex essential for NS5-mediated STAT2 degradation. Depletion of ZSWIM8 confers partial resistance to ZIKV infection in A549 cells, reducing ZIKV-induced cytopathic effects. Notably, in neural progenitor cells, ZSWIM8 depletion enhances interferon signaling, owing to sustained STAT2 levels, thereby inhibiting ZIKV infection. Our findings offer unique insights into the interplay of ZIKV and host, particularly the role of NS5 in manipulating the ZSWIM8–CUL3 E3 ligase complex to orchestrate STAT2 degradation, with implications for developing antiviral strategies and prophylactic interventions.

Abstract

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8–CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8–CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV–host interactions and holds promise for the development of antivirals and prophylactic vaccines.

The Interferon (IFN) system plays a vital role as an innate antiviral defense strategy employed by the host (1, 2). Extensive evidence demonstrates that individuals with genetic defects in the IFN signaling pathway are unable to effectively control even attenuated viruses (3–5), highlighting the essential role of IFNs in immediate intrinsic cellular defenses against viruses. Currently, three distinct types of IFNs, namely types I (IFN-I), IFN-II, and IFN-III, have been identified and classified based on their genetic, functional properties, and receptor usage (6). Among these, IFN-I, characterized by the widespread expression of the surface receptor IFNAR composed of IFNAR1 and IFNAR2 subunits, serves as the primary line of defense against viral infections. The IFN-I family in humans includes IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω (7). Upon pathogen detection, IFN-I is produced and binds to the IFNAR, initiating a cascade through the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway. This cascade leads to the activation of the IFN-stimulated gene (ISG) factor 3 (ISGF3), consisting of phosphorylated STAT1, STAT2, and the IFN regulatory factor 9 (IRF9) (8, 9). ISGF3 binds to the IFN-stimulated response elements (ISRE) in the promoter regions of various genes, triggering the transcriptional activation of hundreds of ISGs. These ISGs collectively contribute to direct antiviral, antiproliferative, and immunoregulatory activities, fortifying the host's defense against viral infections (10, 11).

Viruses have evolved diverse strategies to evade the IFN-I antiviral responses for their replication (1, 12). For instance, the Hepatitis C virus (HCV) encodes several proteins that interfere with IFN-I antiviral responses. The NS3/4A protease cleaves the cellular innate immune adaptors MAVS (VISA/IPS-1/Cardif) (13, 14) and TRIF (15), while the NS4B protein suppresses STING/MITA, resulting in decreased IFN production (16). Similarly, the influenza virus NS1 protein acts as a multifunctional protein that interferes with IFN production and counteracts the antiviral actions of ISGs through various mechanisms (17–19). Specifically, NS1 interacts with TRIM25, an E3 ubiquitin ligase required for RIG-I activation and IFN-I production, thereby dampening the host immune response (20). Additionally, NS1 regulates mRNA production and indirectly inhibits the IFN signaling pathway (21, 22).

Flaviviruses, including Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), Yellow fever virus (YFV), and Japanese encephalitis virus (JEV), are enveloped, positive sense single-stranded RNA viruses with a genome of approximately 11 kb (23, 24). Their genomes consist of a single open reading frame (ORF) flanked by the 5′-untranslated region (UTR) and 3′-UTR. The ORF encodes a polyprotein that undergoes proteolytic cleavage by both host and viral proteases, resulting in three structural proteins (capsid, C; precursor membrane, prM; and envelope, E), and at least seven nonstructural (NS) proteins (NS1, NS2A/B, NS3, NS4A/B, and NS5) (23). Flaviviruses pose severe health risks to humans and animals (25). Among them, ZIKV infection can lead to pregnancy loss and neurological syndromes such as microcephaly and Guillain–Barré syndrome. To date, no prophylactic vaccines or specific antiviral treatments are available to prevent or treat ZIKV infection (26).

Flaviviruses have evolved multiple strategies to subvert IFN-I-mediated restriction. The NS2B3 protease of DENV, ZIKV, JEV, and WNV specifically cleaves human STING (27–29), while the NS5 protein of DENV and ZIKV targets human STAT2 for degradation in a species-specific manner (30, 31). DENV NS5 utilizes the E3 ubiquitin ligase UBR4 to degrade STAT2 (32), whereas ZIKV NS5 achieves STAT2 degradation independently of UBR4 (30). This highlights a convergence among flavivirus NS5 proteins in evading IFN antiviral responses through distinct mechanisms. Given the critical role of STAT2 in IFN-I antiviral signaling, a comprehensive understanding of the mechanisms underlying NS5-mediated STAT2 degradation is essential for unraveling pathogenesis and informing therapeutic approaches. In this study, we aim to elucidate how ZIKV NS5 degrades human STAT2 and investigate the interplay between STAT2 degradation and virus infection.

Results

A Genome-Wide CRISPR/Cas9 Knockout Screen Identifies Essential Host Factors for ZIKV NS5-Mediated STAT2 Degradation.

To provide insights into ZIKV NS5-mediated degradation of human STAT2, we conducted a genome-wide CRISPR/Cas9 knockout (KO) screen to identify the essential host factors involved in this event (33). For this purpose, we engineered HEK293T^{STAT2-mCherry-Flag/rtTA-HA-NS5} reporter cells (hereafter HEK293T reporter cell), which harbored human STAT2-mCherry-Flag under the CMV promoter and a ZIKV HA-NS5 cassette regulated by the Tet-On system (Fig. 1A). Treatment of HEK293T reporter cells with Doxycycline (Dox) induced ZIKV NS5 expression. The subsequent reduction in STAT2-mCherry signal, from 98.4% to 6.87% (Fig. 1 B and C, lanes 3 and 4), along with its rescue by the proteasome inhibitor MG132 (30), validated the functionality of the reporter system (SI Appendix, Fig. S1A, lanes 4 and 6).

Fig. 1.

Identification of host factors essential for NS5-mediated STAT2 degradation by CRISPR/Cas9 screen. (A) Schematic representation of the reporter system monitoring NS5-mediated STAT2 degradation. Components include rtTA (reverse tetracycline-controlled transactivator), TRE (Tetracycline-responsive element promoter), IRES (internal ribosome entry site), and BSD (blasticidin S deaminase). (B and C) The responsiveness of STAT2-mCherry protein to NS5 expression. The HEK293T^{STAT2-mCherry-Flag/rtTA-HA-NS5} reporter cells were treated with or without doxycycline (2 μg/mL) for 3 d. Collected cells underwent flow cytometry and immunoblotting analysis to assess NS5 and STAT2-mCherry expression. (D) Genome-wide CRISPR/Cas9 KO library screening schematic. The Brunello CRISPR/Cas9 KO library was lentivirally introduced into Cas9-overexpressing HEK293T reporter cells (MOI = 0.3). After two rounds of sorting for mCherry-positive cells upon doxycycline treatment, genomic DNA was extracted, and the abundance of sgRNA fragments was assessed via high-throughput sequencing. (E) Enrichment significance plot of host factors identified in the first CRISPR/Cas9 KO screen. Circles represent genes, with the y axis indicating enrichment significance by MAGeCK analysis. The top 10 genes are highlighted. (F) Bar plot of the gene ontology (GO) analysis depicting enriched GO terms for the top 20 hits, analyzed using the Metascape analysis webtool.

Subsequently, the human genome-wide CRISPR/Cas9 KO library (Brunello) targeting 19,114 genes with 76,441 guide RNAs (four sgRNAs per gene) (34) was introduced into the HEK293T reporter cells. After puromycin selection for stable CRISPR viral integration, the resulting cellular library, encompassing approximately 4 × 10⁷ independent viral integration events was treated with Dox to induce ZIKV NS5 expression. Following three days of induction, fluorescence-activated cell sorting (FACS) was employed to isolate the cell populations expressing a high level of mCherry (Fig. 1D), as CRISPR KO of genes essential for NS5-mediated STAT2 degradation would maintain STAT2-mCherry expression. Genomic DNA was extracted and single-guide RNA (sgRNA) sequences were amplified by PCR from the sorted population and unsorted control library cells for deep sequencing to assess the enrichment of each sgRNA in mCherry-positive population. The sorted mCherry-positive cells were allowed to repopulate once, followed by another round of ZIKV NS5 induction and cell sorting (Fig. 1D). The mCherry-positive cells, indicative of genetic KOs resistant to ZIKV NS5-mediated STAT2 degradation, were enriched through two sorting rounds (SI Appendix, Fig. S1B). MAGeCK analysis identified recurring hits (Dataset S1), with the top 20 genes showing stability and effectiveness across two screens (SI Appendix, Fig. S1C). Here, we show the gene enrichment of the first screening as this screen was robust as indicated by the enrichment of multiple individual sgRNAs against the top 10 scoring genes (Fig. 1E and SI Appendix, Fig. S1D). Additionally, GO enrichment analysis of the identified top 20 hits revealed a significant enrichment of genes associated with diverse biological processes, including transcription, organelle assembly, muscle structure development, regulation of catabolic processes, and catalytic activities, as well as protein ubiquitination (Fig. 1F). The top-ranking hits and enriched biological processes collectively highlight potential host factors crucial for ZIKV NS5-mediated STAT2 degradation.

ZSWIM8 Is Required for ZIKV NS5-Mediated STAT2 Degradation.

We selected the top 8 genes with a cutoff of false discovery rate (FDR) <0.02 for validation. For each specific gene target, HEK293T reporter cells were transduced with two independent sgRNAs and a nontargeting sgRNA (sgNC) was also transduced as the control. Upon NS5 induction by Dox, we evaluated STAT2-mCherry expression in the KO cells using flow cytometry. Notably, HEK293T reporter cells transduced with sgRNAs for ZSWIM8, MED16, MED25, STK11, and TCEB2 exhibited high mCherry expression after Dox treatment (Fig. 2A and SI Appendix, Fig. S2A). Western blot (WB) analysis revealed that HA-NS5 induction was significantly weakened after KO of the Mediator subunits MED16 and MED25, leading to inadequate degradation of STAT2-mCherry-Flag (SI Appendix, Fig. S2B, lanes 18, 20, 28, and 30). The Mediator Complex plays an important role in regulating of RNA Polymerase II-mediated gene transcription (35–37). Considering the dependence of the Tet-on-HA-NS5 cassette on PiggyBac transposase (PBase) for random integration in HEK293T reporter cells, and as PBase preferentially inserts transposons near superenhancers regions bound by Mediator complex (38, 39), therefore, depletion of MED16 and MED25 yielded the anticipated results. Additionally, several other genes encoding kinases, including PRKAR1A, SIK2, and STK11, components in LKB1/AMPK pathway, were unexpectedly among the top hits in the screen. Ablation of Protein Kinase CAMP-Dependent Type I Regulatory Subunit Alpha (PRKAR1A), Serine/Threonine Kinase 11 (STK11) and its downstream kinase Salt Inducible Kinase 2 (SIK2) resulted in higher STAT2-mCherry expression at basal level (without NS5 expression) (SI Appendix, Fig. S2B, lanes 7, 9, 21, 23, 31, and 33). Loss of PRKAR1A and STK11 conferred resistance to NS5-mediated STAT2 degradation in reporter cells, suggesting their involvement in modulating STAT2 protein modification and stability (Fig. 2A). However, in cells lacking TCEB2 (also known as Elongin B, ELOB) and the less well-characterized protein Zinc finger SWIM domain-containing protein 8 (ZSWIM8), the efficiency of Dox-induced HA-NS5 expression was not significantly affected compared to sgNC cells. Flow cytometry results showed that STAT2-mCherry remained above 70% after Dox induction (Fig. 2A). Furthermore, these two genes fall into the components of Cullin-RING E3 ligase (CRL) ligase and are involved in protein ubiquitination pathway (Fig. 1F). This suggested that our screen uncovered the key components of an E3 ubiquitin ligase crucial for ZIKV NS5-mediated STAT2 degradation.

Fig. 2.

Validation of ZSWIM8 function in NS5-mediated STAT2 degradation. (A) The host factors as indicated were knocked out by CRISPR/Cas9 in HEK293T reporter cells, and then STAT2-mCherry expression was analyzed by flow cytometry after 48 h of 2 μg/mL Dox treatment. A nontargeting sgRNA (sgNC) served as control. (B) The plasmids of HA-NS5 and/or STAT2-Flag were transfected into the HEK293T transduced with sgNC or sgRNA targeting ZSWIM8. After 2 d, cells were collected for immunoblotting assay to detect NS5 and STAT2 expression. (C) Huh7 cells were lentivially transduced with sgRNA targeting ZSWIM8, URB4 genes, or sgNC, followed by puromycin selection to generate KO cells. The cells were then infected with ZIKV (SPH2015 strain, MOI = 0.2, Upper panel) or DENV (DENV-2 16681 strain, MOI = 0.2, Lower panel). After 36 h of infection, the cells were subjected to immunoblotting assays to assess STAT2, STAT1, viral NS5, and actin levels. (D) HEK293T sgNC and ZSWIM8 KO cells were complemented with vector or sgRNA-resistant Flag-ZSWIM8, which were transiently cotransfected with HA-NS5 plasmids. Immunoblotting analysis was performed to detect the abundance of STAT2 and other proteins. (E) HEK293T reporter cells and the ZSWIM8 overexpressed reporter cells were treated with 2 μg/mL Dox. At the indicated time points, cells were analyzed by flow cytometry to determine the percentage of STAT2-mCherry in WT and ZIWIM8-overexpressed cells. (A and E) This experiment was independently repeated three times and the representative data are presented as mean ± SD (n = 3). (B–D) The experiment was independently repeated three times, and the representative images are displayed.

We directed our efforts to further dissect the role of ZSWIM8 in ZIKV NS5-mediated STAT2 degradation. We generated individual HEK293T KO cells using two sgRNAs for ZSWIM8 and transiently expressed STAT2 and NS5. Remarkably, ZSWIM8 depletion significantly rescued STAT2 protein levels compared to cells with sgNC cells (Fig. 2B, lanes 4 and 6). This finding was corroborated by ZIKV (MR766 and PRVABC59 strains) infection in Huh7 ZSWIM8 KO cells and sgNC cells (SI Appendix, Fig. S3 A, Right panel, lanes 2 and 6), confirming ZSWIM8-dependent degradation of STAT2 by ZIKV NS5. To rule out potential cell-line-specific effects, we conducted ZIKV infections in A549 ZSWIM8 KO cells, yielding consistent results (SI Appendix, Fig. S3 B, Right panel, lanes 4 and 6). Previous studies had demonstrated that the E3 ubiquitin ligase UBR4 is recruited by DENV NS5 for STAT2 degradation (32). Therefore, we examined whether the ZSWIM8 plays a specific role in STAT2 degradation by ZIKV NS5. To assess this, we separately knocked out UBR4 and ZSWIM8 in Huh7 cells using CRISPR/Cas9 technique, and then the Huh7 URB4 KO and ZSWIM8 KO cells were infected with ZIKV or DENV-2, respectively. Notably, UBR4 KO did not rescue STAT2 protein levels after ZIKV infection (Fig. 2 C, Upper panel, lanes 6 and 8), and ZSWIM8 KO did not restore STAT2 degradation in DENV-infected cells (Fig. 2 C, Lower panel, lanes 2 and 4). This is consistent with previous observations that ZIKV NS5 does not interact with the UBR4 ligase (30), suggesting a virus-specific mechanism in NS5-mediated STAT2 degradation.

To further confirm that the rescue of STAT2 was the result of ZSWIM8 loss, we reconstituted ZSWIM8 KO cells with cDNAs expressing CRISPR sgRNA-resistant versions of ZSWIM8 or an empty vector as a control. ZSWIM8 overexpression efficiently restored NS5-induced STAT2 degradation in both HEK293T cells (Fig. 2D, lanes 8 and 10) and reporter cells (SI Appendix, Fig. S3C). Furthermore, ZSWIM8 overexpression in HEK293T reporter cells significantly accelerated STAT2-mCherry degradation compared to that in vector-transfected reporter cells under the same Dox treatment time (Fig. 2E). This result was further supported by cotransfection of NS5, STAT2, and ZSWIM8 in HEK293T cells (SI Appendix, Fig. S3D, lane 3). Collectively, these results demonstrate the crucial role of ZSWIM8 in ZIKV NS5-induced STAT2 degradation.

ZSWIM8 Functions as the Substrate Receptor of CUL3, Mediating STAT2 Degradation by ZIKV NS5.

ZSWIM8 contains BC-box and Cullin-box motif in its N-terminal region, facilitating interaction with Elongin B (ELOB/TCEB2) and Cullin proteins, respectively (40), forming a substrate adaptor of the CRL (Fig. 3A). Previously implicated in target-directed microRNA degradation (TDMD) (41, 42), the ZSWIM8/Cullin 3-RING ligase complex plays diverse roles, such as mediating the degradation of misfolded SAX-3/Robo receptors in Caenorhabditis elegans to ensure accurate axon guidance (43). CRLs, the largest subfamily responsible for protein ubiquitination, consist of Cullin scaffold proteins, adaptor, and substrate receptor (44). CRL activation necessitates Cullins neddylation, a process involving the conjugation of NEDD8 to Cullin proteins (45, 46). To investigate whether the Cullin proteins are involved in the STAT2 degradation by ZIKV NS5, we used the small molecule MLN4924, a Cullin neddylation inhibitor (47), to treat HEK293T cells transfected with NS5 and STAT2. WB results showed that the neddylation level of Cullin 3 (CUL3, a member of Cullin family) was decreased by MLN4924 treatment, accompanied by a significant restoration of STAT2 protein levels (Fig. 3B, lane 6). Consistent results were observed in HEK293T reporter cells, where MLN4924 treatment, in the presence of Dox, resulted in the rescue of STAT2-mCherry levels (Fig. 3C). These findings indicate that ZIKV NS5 targets STAT2 for degradation through the recruitment of the CRL.

Fig. 3.

ZSWIM8–CUL3 E3 ubiquitin ligase requirement for ZIKV NS5-mediated STAT2 degradation. (A) Components of the ZSWIM8 Cullin-RING ubiquitin ligase. Key components include MLN4924 (NEDD8-activating enzyme E1 inhibitor), NAE1 (NEDD8-activating enzyme E1 subunit 1), N8 (Nedd8 modification), EloB (elongin B), E2 (ubiquitin-conjugating enzyme), Ub (ubiquitin), SWIM (SWI2/SNF2 and MuDR domain), and A (conserved domain A). (B) HEK293T cells were transfected with STAT2-Flag and HA-NS5 plasmids. At 36 h posttransfection, the cells were treated with 10 μM MG132 or 1 μM MLN4924 for 12 h. Immunoblotting assessed STAT2 and NS5 levels, with CUL3 indicating MLN4924 efficacy. (C) The HEK293T reporter cells treated with 2 μg/mL Dox for 24 h, followed by varying doses of MLN4924 (1, 3, or 10 μM) treatment for another 12 h. Flow cytometry analyzed STAT2-mCherry percentage. (D) Cullin genes as indicated were knocked out in the HEK293T reporter cells, Flow cytometry assessed STAT2-mCherry after 48 h of 2 µg/mL Dox treatment. (E) Huh7 cells, transduced with sgRNA targeting CUL2, CUL3, and CUL5, were infected with ZIKV (MOI = 0.2), and then the cells were collected at 36 h postinfection for an immunoblotting assay to assess expression levels of STAT2 and viral NS5 protein. (F) HEK293T cells were cotransfected with plasmids expressing STAT2-Flag, ZIKV NS5, and HA-ZSWIM8 or Myc-CUL3 in various combinations as indicated. After 36 h, 10 μM MG132 was added for an additional 10 h, followed by cell collection and lysis. Immunoprecipitation was performed using Flag antibody-conjugated magnetic beads, and immunoprecipitants were subjected to immunoblotting analysis to examine STAT2 ubiquitination. (G) HEK293T cells were transfected with plasmids expressing Myc-CUL3 and either Flag-ZSWIM8-FL or Flag-ZSWIM8-ΔBox. After 48 h, cell lysates were immunoprecipitated using Flag antibody-conjugated magnetic beads. The immunoprecipitants were subsequently analyzed by immunoblotting assay using Flag and Myc antibodies. (B and E–G) The experiment was independently repeated three times and the representative images are displayed. (C and D) This experiment was independently repeated three times and the representative data are presented as mean ± SD (n = 3).

We next investigated the role of different members of the Cullin family in NS5-induced STAT2 degradation through genetic depletion using CRISPR/Cas9 techniques. To this end, we generated individual KO cells in HEK293T reporter cells targeting CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and notably, only CUL3 KO resulted in a significant rescue of STAT2-mCherry levels (Fig. 3D and SI Appendix, Fig. S4A). Importantly, the efficiency of NS5 induction remained unaffected by depletion of Cullin proteins (SI Appendix, Fig. S4B). These data highlight the putative contribution of the CUL3 E3 ligase to STAT2 degradation by ZIKV NS5. The ZSWIM8/CUL3 E3 ligase complex model includes ELOB/ELOC (EloBC) as substrate adaptors (42), consistent with our observation that ELOB/TCEB2 was a top hit in the initial screen (Fig. 2A). To further verify the specificity of CUL3 in STAT2 degradation compared to other Cullin proteins under viral infection conditions, we assessed STAT2 levels in Huh7 cells depleted of CUL2, CUL3, and CUL5 during ZIKV infection. Our results demonstrated that CUL3 depletion significantly rescued STAT2 protein levels upon ZIKV infection. In contrast, depletion of CUL2 and CUL5 had no discernible impact on STAT2 degradation, suggesting a CUL3-dependent mechanism in STAT2 degradation induced by ZIKV infection (Fig. 3E, lanes 4 and 6). Given the pivotal role of E3 ligases in substrate ubiquitination, we then examined the ubiquitination level of STAT2. Ubiquitination of STAT2 was observed in both vector and ZIKV NS5 overexpressed cells, indicating the involvement of the ubiquitin–proteasome system in STAT2 regulation. Importantly, overexpression of CUL3 and ZSWIM8 led to a 1.4-fold increase in STAT2 ubiquitination (Fig. 3F, lane 4), highlighting the role of CUL3-mediated ubiquitination in regulating STAT2 stability in the presence of ZIKV NS5.

We proceeded to determine the binding of ZSWIM8 to CUL3 by generating a truncation with the deletion of the BC-box and Cullin-box motif. The immunoprecipitation assay revealed that removal of the N terminus (ZSWIM8–ΔBox) diminished its binding to CUL3, while leaving the interaction with ZIKV NS5 protein unaffected (Fig. 3G, lane 3). In line with this, ectopic expression of WT ZSWIM8 in ZSWIM8 KO cells, but not its mutant ZSWIM8–ΔBox, led to a reduction in STAT2 levels (SI Appendix, Fig. S4C). These findings collectively established that the interaction between ZSWIM8 and CUL3 E3 ligase is required for ZIKV NS5 mediated-STAT2 degradation.

ZIKV NS5 Serves as a Scaffold Protein to Connect ZSWIM8/CUL3 Ligase Complex and STAT2.

Our data supported that ZSWIM8/CUL3 complex is required for ZIKV NS5-mediated STAT2 degradation. We hypothesized that ZSWIM8–CUL3 ligase complex is recruited by the ZIKV NS5 to target STAT2. To test this, we examined the interaction between ZSWIM8 and STAT2 in the presence and absence of NS5 protein through immunoprecipitation assays using HEK293T cells transiently overexpressing Flag-ZSWIM8, STAT2 and NS5. In the absence of NS5, there was a limited level of coprecipitated STAT2 with Flag-ZSWIM8 (Fig. 4A, lane 6). However, NS5 overexpression significantly increased the binding of ZSWIM8 to STAT2, even though NS5 expression caused a reduction in STAT2 levels (Fig. 4A, lane 7), and this interaction was further enhanced after STAT2 levels were restored by MG132 treatment (Fig. 4A, lane 8). In addition, immunoprecipitation assays using HEK293T cells transfected with HA-NS5, along with individual expression vectors for STAT2, ZSWIM8, and CUL3, revealed that NS5 coprecipitated with the ZSWIM8/CUL3 ligase complex as well as with STAT2 (Fig. 4B). Moreover, the interaction between NS5 and CUL3 was slightly enhanced with exogenously expressed ZSWIM8, which is consistent with the role of ZSWIM8 as a substrate receptor of CUL3 (Fig. 4B, lanes 2 and 5). Considering that the ZSWIM8 N terminus is responsible for CUL3 interaction, we speculated that the remaining region of ZSWIM8 is involving in binding NS5. To test this, we constructed a ZSWIM8 N-terminal (1–231aa) truncation including the BC-box, Cullin-box, and SWIM domain, as well as a ZSWIM8 C-terminal (232–1834aa) truncation mutant (SI Appendix, Fig. S5A), and then assessed their interaction with NS5. WT ZSWIM8 and mutants retaining the C-terminal region interacted with NS5 (SI Appendix, Fig. S5B). However, each mutant, when expressed, was insufficient for STAT2 degradation, but exerted a dominant-negative effect that efficiently restored STAT2 levels in HEK293T reporter cells following Dox treatment (SI Appendix, Fig. S5C).

Fig. 4.

ZIKV NS5 enhances the interaction of ZSWIM8 and STAT2. (A) HEK293T cells were cotransfected with plasmids expressing Flag-ZSWIM8, STAT2, or ZIKV NS5. After 36 h, one set of the cells was treated with 10 μM MG132 for an additional 10 h. Subsequently, cells were collected, lysed, and subjected to immunoprecipitation using Flag antibody-conjugated magnetic beads. Immunoprecipitants were analyzed by immunoblotting with Flag, NS5, and STAT2 antibodies. (B) HEK293T cells were cotransfected with the plasmids expressing HA-NS5, Flag-ZSWIM8, STAT2, and Myc-CUL3. After 48 h, cells were collected and lysed. Immunoprecipitation was performed using HA antibody-conjugated magnetic beads, and immunoprecipitants were analyzed by immunoblotting. (C) HEK293T sgNC cells were transfected with Flag-ZSWIM8 and/or NS5. HEK293T STAT2 KO cells were transfected with Flag-ZSWIM8, NS5, and complemented with STAT2 expression. After 48 h, cells were collected and lysed. Immunoprecipitation was carried out using Flag antibody-conjugated magnetic beads, and immunoprecipitants were subjected to immunoblotting analysis. (D) HEK293T sgNC cells and ZSWIM8 KO cells were transfected with NS5 and STAT2-Flag plasmids. After 48 h, cells were collected and lysed. Immunoprecipitation was conducted using Flag antibody-conjugated magnetic beads, and the immunoprecipitants were analyzed by immunoblotting. (E) Proposed model illustrating the ZSWIM8 Cullin-RING ubiquitin ligase mediating STAT2 degradation by ZIKV NS5. All the experiment were independently repeated three times and the representative images are displayed.

To further explore whether the interaction between NS5 and ZSWIM8 is dependent on substrate recognition, we performed immunoprecipitation assays in HEK293T sgNC and STAT2 KO cells by cotransfecting Flag-ZSWIM8 with ZIKV NS5. As shown in Fig. 4C, ZIKV NS5 could be coprecipitated with Flag-ZSWIM8 in HEK293T sgNC cells (Fig. 4C, lane 5). The absence of detectable STAT2 protein in HEK293T sgNC lysates may be attributed to NS5-mediated degradation (Fig. 4C, lanes 4 and 5). Importantly, the depletion of STAT2 did not disrupt the binding between ZSWIM8 and NS5, and this interaction remained unaffected by exogenously expressed STAT2 (Fig. 4C, lanes 2 and 3). These results suggest that ZIKV NS5 plays a pivotal role in connecting ZSWIM8 and STAT2. Furthermore, NS5 retained its ability to bind STAT2 in ZSWIM8-depleted cells (Fig. 4D, lane 2), providing the prerequisite for STAT2 degradation. Taken together, our results propose that NS5 acts as the scaffold protein, orchestrating the ZSWIM8/CUL3 E3 ligase complex to target STAT2 for degradation (Fig. 4E). ZSWIM8 functions as an adaptor protein to mediate CUL3 interaction and NS5 recognition through its N-terminal and C-terminal domains, respectively (Figs. 3G and 4 B and E). These coordinated processes facilitate the degradation of STAT2 during ZIKV infection.

Genetic Depletion of ZSWIM8 Confers Resistance of the Cells to ZIKV Infection.

After confirming the biochemical interaction between the ZSWIM8/CUL3 complex, NS5 and STAT2, we sought to evaluate the functional significance of STAT2 degradation. ZIKV infection in Huh7 sgNC and Huh7 ZSWIM8 KO cells revealed that ZSWIM8 depletion restored STAT2 levels, and ZIKV infection and grow kinetics were significantly reduced in ZSWIM8 KO cells, indicating impaired ZIKV infection (Fig. 5 A and B and SI Appendix, Fig. S6A). To further elucidate the impact of ZSWIM8 on ZIKV infection, we knocked out ZSWIM8 in A549 cells, a human lung epithelial cancer cell line with functional IFN signaling. Subsequent ZIKV (SPH2015 strain) infection assay revealed a marked reduction of ZIKV infection in A549 ZSWIM8 KO cells, an effect that was reversed upon additional STAT2 KO (A549 ZSWIM8/STAT2 double KO, DKO) (Fig. 5 C and D).

Fig. 5.

Protective role of ZSWIM8 loss against ZIKV infection. (A and B) Huh7 cells were transduced with sgRNA targeting ZSWIM8 or sgNC and infected with ZIKV at the MOI of 1. After 24 h, cells were fixed, stained for E (green), STAT2 (red), and nuclei (blue). White arrows indicate instances of STAT2 degradation in ZIKV-infected Huh7 cells. Fluorescence plot profiles (at dashed lines) were analyzed using ImageJ. Infection rates were determined by counting ZIKV E-positive cells as a percentage of total cells. Data are presented as mean ± SD. Statistical significance was assessed using two-tailed unpaired t test (***P < 0.001). (Scale bar, 10 μm.) (C) A549 cells were transduced with sgRNA targeting ZSWIM8. ZSWIM8 KO cells were subsequently transduced with STAT2 sgRNA lentivirus, generating ZSWIM8/STAT2 DKO cells. Whole-cell lysates were analyzed by immunoblotting assay to assess the ZSWIM8 and STAT2 levels. (D) A549 ZSWIM8 KO and ZSWIM8/STAT2 DKO cells, along with the control (sgNC), were infected with ZIKV at the MOI of 0.05. Flow cytometry analysis at 48 h and 60 h postinfection determined the percentage of ZIKV-infected cells. Statistical significance was determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). (E and F) A549 sgNC, A549 ZSWIM8 KO cells, and ZSWIM8/STAT2 double KO cells were infected with ZIKV (MR766) at an MOI of 0.01. Cell culture medium was collected at indicated time point to determine the virus titer. Results are represented as a mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA (ns, no significance; *P < 0.05; ***P < 0.001). After 7 d of infection, cells were fixed, stained with crystal violet, and observed for survival cells. This experiment was independently repeated three times with similar results and the representative data are presented as mean ± SD (n = 3). (G) A549 sgNC or ZSWIM8 KO cells were infected with ZIKV at the MOI of 1. After 24 h, cells were collected for RNA extraction and subsequent transcriptomic analysis. The heatmap was generated by calculating |Log2FC| of up-regulated genes enriched in immune response, comparing infected and mock cells.

We further analyzed ZIKV growth kinetics and observed a significant reduction in ZIKV titers (MR766 strain), approximately 12-fold lower at 72 h postinfection in A549 ZSWIM8 KO cells compared to A549 sgNC and ZSWIM8/STAT2 DKO cells. The difference in ZIKV titers between A549 ZSWIM8KO and sgNC or ZSWIM8/STAT2 DKO cells at 96 h was comparatively smaller, likely attributable to the saturation of ZIKV replication at later stages. Notably, in A549 ZSWIM8/STAT2 DKO cells, ZIKV exhibited similar growth kinetics as in A549 sgNC at all observed time points (Fig. 5E), suggesting that the reduced infectivity of ZIKV in A549 ZSWIM8 KO cells is due to the inability of ZIKV NS5 to degrade STAT2 protein in the absence of ZSWIM8. Consistent with the growth kinetics, the surviving cells visibly increased in ZSWIM8-depleted A549 cells after ZIKV infection compared to sgNC and ZSWIM8/STAT2 DKO cells (Fig. 5F), indicating that ZSWIM8 KO protected A549 cells from the ZIKV-induced cytopathic effect. We further assessed the growth kinetics of the ZIKV SPH2015 strain in A549 sgNC, A549 ZSWIM8 KO, and A549 ZSWIM8/STAT2 DKO cells. In line with the observations for the MR766 strain, we noticed a significant reduction in ZIKV titers (SPH2015 strain) in A549 ZSWIM8 KO cells, compared to A549 sgNC and ZSWIM8/STAT2 DKO cells. Similar to the findings with the MR766 strain, ZIKV exhibited comparable growth kinetics in A549 sgNC and ZSWIM8/STAT2 DKO cells (SI Appendix, Fig. S6B). Following ZIKV SPH2015 infection, A549 ZSWIM8 KO cells exhibited greater survival compared to sgNC and ZSWIM8/STAT2 DKO cells (SI Appendix, Fig. S6C), indicating that ZSWIM8 KO also provided protection against the cytopathic effect induced by the ZIKV SPH2015 strain. Furthermore, ZSWIM8 does not play a direct role in the regulation of IFN and ISGs expression, as the KO of ZSWIM8 did not impact the expression of IFN and ISGs after poly(I:C) stimulation (SI Appendix, Fig. S6D). To further establish that the impaired viral growth kinetics in A549 ZSWIM8 KO cells was due to the activation of IFN signaling, we compare the viral grow kinetics in A549 sgNC and ZSWIM8 KO cells in the presence or absence of ruxolitinib (2 μM), which is a potent and selective inhibitor of Janus kinase (JAK)1 and JAK2, could inhibit JAK-STAT pathway to prevent IFN signaling (48, 49). It is shown that compared to A549 sgNC cells, viral titer was dramatically reduced after 48 h in A549 ZSWIM8 KO cells. However, the viral titer in cells treated with ruxolitinib was significantly higher than in untreated cells regardless of ZSWIM8 depletion (SI Appendix, Fig. S6E). This suggests that the reduced viral infectivity in A549 ZSWIM8 KO cells was due to the activation of IFN signaling. Taken together, these results suggest that ZSWIM8 depletion delays ZIKV growth kinetics at later stages postinfection, and this effect appears to be related to the inability of ZIKV NS5 to degrade STAT2, leading to the activation of IFN signaling in the absence of ZSWIM8.

To confirm that the growth reduction of ZIKV in ZSWIM8 depletion cells was due to the restoration of STAT2 and enhanced activation of IFN signaling, we then analyzed the transcriptomic profiles by RNA sequencing (RNA-seq) in A549 ZSWIM8 KO cell after ZIKV infection. Differentially expressed genes (DEGs) analysis revealed that ZIKV infection induced dramatic gene expression changes in A549 sgNC and ZSWIM8 KO cells, compared with the uninfected counterpart cells. Of note, a cluster of ISGs and immune response-related genes exhibited a more pronounced upregulation in A549 ZSWIM8 KO cells compared to sgNC cells (Fig. 5G). These findings were further validated by RT-qPCR, which demonstrated a significant increase in the expression levels of IFN-β and specific ISGs, such as IFITM3, ISG15, MxA, ISG56, and RIG-I, in A549 ZSWIM8 KO cells compared to sgNC cells (SI Appendix, Fig. S6F). Overall, these results suggest that ZSWIM8 depletion allows for the restoration of STAT2 and the activation of IFN signaling, thereby establishing an antiviral state against ZIKV infection.

ZSWIM8 Is Involved in ZIKV NS5 Antagonism of IFN Signaling in Neural Progenitor Cells.

ZIKV exhibits specific neurotropism and has been implicated in inducing neurological disorders (50, 51). ZIKV targets various cells in the brain, with human neural progenitor cells (hNPCs) being the primary focus during infection (52, 53). To investigate the relevance of ZSWIM8 in IFN antagonism by ZIKV in a physiologically relevant model, we constructed ZSWIM8 KO cells using hNPCs and examined ZIKV infection. In hNPCs infected with MR766 or SPH2015 strain, a notable reduction in STAT2 levels was observed (Fig. 6A, lane 2; Fig. 6B, lane 2). Crucially, the degradation of STAT2 induced by ZIKV infection was significantly rescued by genetic loss of ZSWIM8 (Fig. 6A, lanes 4 and 6; Fig. 6B, lanes 4 and 6). To assess the effect of restored STAT2 levels on ZIKV infection, we challenged the hNPCs sgNC and ZSWIM8 KO cells with ZIKV. As expected, the virus infection was decreased in ZSWIM8-depleted cells (Fig. 6C). Next, we evaluated ZIKV growth kinetics in sgNC, ZSWIM8 KO, and ZSWIM8/STAT2 DKO hNPCs (Fig. 6D). Viral titers in ZSWIM8 KO NPCs were markedly reduced compared to those in sgNC and ZSWIM8/STAT2 DKO cells (Fig. 6E). However, in ZSWIM8/STAT2 DKO cells, ZIKV titer was even higher than those in sgNC cells at 72 h post infection (Fig. 6E), indicating that the impaired infectivity of ZIKV in ZSWIM8 KO NPCs was dependent on the retained STAT2 protein.

Fig. 6.

ZSWIM8 involved in ZIKV NS5 antagonism of IFN signaling in hNPCs. (A and B) hNPCs were transduced with ZSWIM8-targeting sgRNA or sgNC and infected with ZIKV MR766 (MOI = 1) or SPH2015 (MOI = 2). After 24 h, cells were collected for immunoblotting. (C) hNPCs sgNC and hNPCs ZSWIM8 KO were infected with ZIKV MR766 at the MOI of 1. After 24 h, cells were fixed and stained for E (green) and nuclei (blue). Infection rate was quantified by counting the ZIKV E-positive cells as a percentage of total cells. Data are presented as mean ± SD. Statistical significance was determined using a two-tailed unpaired t test (***P < 0.001). (Scale bar, 20 μm.) (D) hNPCs ZSWIM8 KO were transduced with STAT2-targeting sgRNA to generate the ZSWIM8/STAT2 DKO cells. Whole-cell lysate was analyzed by immunoblotting to detect ZSWIM8 and STAT2 levels. (A–D) This experiment was independently repeated three times and representative images are shown. (E) hNPC sgNC, ZSWIM8 KO cells, and ZSWIM8/STAT2 DKO cells were infected with ZIKV (MOI = 0.05). Virus titers were determined by collecting cell culture medium at indicated time points. This experiment was independently repeated three times with similar results and the representative data are presented as a mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA (*P < 0.05; **P < 0.01). (F) hNPC sgNC and ZSWIM8 KO cells were infected with ZIKV (MOI = 1). After 24 h, cells were collected for RNA extraction and transcriptomic analysis. The heat map was generated by calculating |Log2 FC| of up-regulated genes enriched in immune response from infected and uninfected (mock) cells.

To elucidate the mechanism restricting ZIKV infection in ZSWIM8-depleted NPCs, we preformed transcriptomic analysis of the infected cells. Hundreds of DEGs were identified in both sgNC and ZSWIM8 KO hNPCs upon ZIKV infection. Remarkably, the expression of ISGs and genes associated with the immune response was notably up-regulated in ZSWIM8 KO hNPCs (Fig. 6F). These results were corroborated by RT-qPCR, revealing a more significant upregulation of specific ISGs, including MDA5, MxA, IFITM1, IFIT1, IFI6, and ISG15 (SI Appendix, Fig. S7). These findings imply that ZSWIM8 depletion, preventing ZIKV-induced STAT2 degradation, leads to a heightened IFN signaling upon ZIKV infection in hNPCs, thereby restricting ZIKV infection.

Discussion

The IFN response stands as one of the primary lines of defense system against flavivirus infections, yet these viruses have evolved diverse strategies to evade IFN signaling. Notably, both DENV and ZIKV utilize the viral protein NS5 to antagonize IFN responses through the proteasome-dependent degradation of STAT2, albeit with distinct mechanisms. DENV NS5 utilized the E3 ubiquitin ligase UBR4 for STAT2 degradation, while this study reveals that ZIKV NS5 exploits ZSWIM8, acting as a substrate adaptor of CUL3-RING E3 ligase, for human STAT2 degradation. Our results underscore the pivotal role of ZIKV NS5 in orchestrating the ZSWIM8–CUL3 E3 ligase complex to interact with and degrade STAT2, promoting efficient ZIKV infection (Fig. 7).

Fig. 7.

Model illustrating ZIKV NS5-mediated reprogramming of the ZSWIM8–CRL3 E3 complex for STAT2 degradation. ZIKV NS5 bridges the ZSWIM8–RL3 complex and STAT2, thereby enhancing their interaction. This interaction promotes the degradation of STAT2 by the proteasome. Depleting ZSWIM8 prevents the degradation of STAT2, resulting in a more pronounced upregulation of genes related to immune response and suppression of ZIKV infection. This underscores the critical role of NS5-mediated STAT2 degradation in dampening IFN signaling and facilitating ZIKV infection.

ZIKV NS5 acts as a scaffold protein, mediating the interaction between ZSWIM8–CUL3 E3 ligase and hSTAT2 (Fig. 4A). Structural studies of ZIKV NS5 and hSTAT2 shown similarities with the DENV NS5–hSTAT2 complex (54, 55), suggesting that while the binding modes are comparable, different host proteins are selected for promoting STAT2 degradation. Therefore, unraveling this intricate interplay is crucial in the future study, and structural approaches like cryo-electron microscopy hold promise in dissecting the NS5-STAT2 and ZSWIM8–CRL3 complex, providing insights into the molecular events driving STAT2 degradation and targeting interactions between NS5, STAT2, and ZSWIM8 could provide a unique antiviral approach.

Moreover, our current understanding of the biological and pathological roles of NS5-mediated degradation of other host proteins remains limited. The unaffected interaction between ZIKV NS5 and ZSWIM8 in the absence of substrate STAT2 (Fig. 4C) implies a role in regulating host protein turnover. Hence, future investigations are necessary to comprehensively identify the substrates targeted by NS5–ZSWIM8 complex. Previous studies show that UBR4, serving as a binder for DENV NS5, not only degrades STAT2 but also ERC1, a regulatory subunit involved in the NF-κB activation pathway (32, 56). It is plausible that the degradation of host proteins may disrupt cellular signaling and function, ultimately affecting tissue physiology and host homeostasis.

Transcriptomic profiling of ZIKV-infected NPCs reveals upregulated immune gene clusters and stronger ISGs induction in ZSWIM8 KO NPCs (Fig. 6F and SI Appendix, Fig. S7). While strain-specific differences remain unexplored, considering the distinctive responses induced by Asian and American/Brazil ZIKV strains, understanding the transcriptional and protein profiles in ZSWIM8-depleted NPCs with different strains is crucial. Although we did not assess the gene transcriptome of NPCs infected by the Asian strain SPH2015, the putative diminished capacity of SPH2015 strain to degrade STAT2 may induce a more significant immune response (Fig. 6 A and B). Compared with the African strain, ZIKV Asian strains have been shown to induce faster and more intensive antiviral responses in brain (57, 58). Interestingly, previous results show that the ZIKV/Brazil strain (American lineage), which is highly associated with Congenital Zika Syndrome (CZS), resulted in a diminished and delayed induction of ISGs and less severe pathological outcomes than the ancestral Asian strain, rendering these strains increased capacity to cross the placenta or to invade the CNS (59, 60). Thus, the transcriptional and protein profiles of ZSWIM8 depleted NPCs infected with Asian or American/Brazil strains are required to deepen our understanding of the strain specific differences.

Remarkably, other flaviviruses such as WNV, Tick-borne encephalitis virus (TBEV), and YFV could antagonize IFN-I pathway using their NS5 proteins (61, 62). For instance, the NS5 proteins of WNV and TBEV interact with prolidase (PEPD), a cellular protein essential for IFNAR1 maturation, which led to disrupting surface expression of IFNAR and thereby inhibiting ISG expression (63). Additionally, TBEV NS5 can also bind to tyrosine kinase 2 (TYK2), which is responsible for the phosphorylation of STAT1 and STAT2 upon IFN-I engagement with its receptor (64), thus inhibiting the transcriptional activation of ISGs (65). YFV NS5 protein binds STAT2 to prevent ISGF3 engagement with ISRE after IFN stimulation, and this binding does not mediate STAT2 degradation (66). These observations suggest that flaviviruses have evolved distinct mechanisms to evade IFN-I singling, highlighting the critical role of IFN-I signaling in control of these viruses. Unraveling these mechanisms is imperative for designing effective antiviral strategies. In conclusion, this study elucidates the ZIKV NS5-mediated hijacking of the ZSWIM8–CRL3 complex as a potent “degrader” of STAT2, emphasizing its critical role in modulating the IFN-I response during ZIKV infection. These findings not only advance our understanding of viral–host interactions but also provide a foundation for therapeutic interventions against flaviviruses.

Materials and Methods

Cell Culture.

HEK293T, A549, Huh7, and Vero cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, China) supplemented with 10% fetal bovine serum (FBS) and 50 IU/mL penicillin/streptomycin in a humidified 5% CO₂ incubator at 37 °C. The hNPC lines were validated using standardized methods as previously described (67). The hiPSC lines were derived from fibroblasts collected from subjects who provided informed consent. To obtain neural progenitor cells, embryoid bodies were formed by mechanically dissociating iPSC colonies and plating them onto laminin-coated dishes. Detailed information on hNPCs generation has been previously reported (67). All samples were deidentified prior to use in this study. All experimental procedures were approved by the Ethics Committee of Tsinghua University (20140081). The hNPCs were maintained in DMEM/F12 (Invitrogen) supplemented with 1% N2, 2% B27, 1% Glutamax, and 20 ng/mL FGF-2, in matrigel-coated dishes at 37 °C with 5% CO₂. All cell lines underwent regular testing to confirm the absence of Mycoplasma contamination.

Statistical Analysis.

Statistical analysis was performed using GraphPad Prism version 8. The data underwent analysis via Student’s unpaired t test or ANOVA as applicable. A significance level of P < 0.05 was deemed statistically significant. Results are presented as means ± SD.

A full overview of the methods and materials referred to in this study is available in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Data, Materials, and Software Availability

RNA-seq data of A549 cells and NPCs have been deposited in the NCBI BioProject repository (accession number PRJNA1141424 and PRJNA1141425) (68, 69). All study data are included in the article and/or supporting information.