TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain–Barré syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target.


Introduction
Zika virus (ZIKV) is a mosquito-borne enveloped, positive-strand RNA virus in the genus Flavivirus and the family Flaviviridae [1]. ZIKV is primarily transmitted to mammals via the bite of an Aedes aegypti mosquito [2]. While symptoms are generally mild in most individuals, ZIKV infection of pregnant women can cause intrauterine growth restriction and microcephaly [3]. As no licensed vaccine or specific antiviral treatment is available, understanding the molecular mechanisms of ZIKV infection is crucial to develop countermeasures [4,5].
The flavivirus RNA genome encodes three structural (capsid, premembrane, and envelope) and seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), with untranslated regions (UTR) flanking the 5 and 3 ends [6]. ZIKV nonstructural proteins play roles in viral replication and assembly [7]. NS1 is a highly conserved nonstructural protein among the flaviviruses, which possesses multiple functions in the viral life cycle, including viral replication, immune evasion, and pathogenesis [8]. A previous study reported that an attenuated recombinant vesicular stomatitis virus-based vaccine expressing ZIKV prM-E-NS1 induces ZIKV-specific antibody and T cell immune responses, providing protection against ZIKV challenge [9]. Monoclonal antibodies targeting NS1 protein can also protect against disease and death in a murine model [10]. These studies suggest that ZIKV NS1 could be a potential therapeutic target.
Here, we utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify host factors that interact with the ZIKV NS1 protein. Among the putative interactions, we

Generation of Stable Cell Lines Suppressing CCT2
To generate cell lines that stably suppress CCT2 protein, Hela cells were transfected with CCT2 MISSION shRNA Plasmid (Thermo Fisher, Branchburg, NJ, USA). Cell clones were selected in DMEM supplemented with 10% (vol/vol) fetal calf serum (FCS), penicillin G (100,000 U/L), streptomycin (100 mg/L), and puromycin (2 µg/mL). Immunoblot was performed to confirm that the expression of CCT2 was suppressed in a selected clone.

Gene Silencing in Mosquitoes
Double-stranded (ds) RNA targeting either a 400 bp region of the A. aegypti CCT2 gene or an irrelevant green fluorescent protein (GFP) gene were transcribed using gene-specific primers designed with a T7 promoter and the MEGAScript RNAi kit (Thermo Fisher Scientific, Ambion, Branchburg, NJ, USA). DsRNA was produced using TranscriptAid T7 High Yield Transcription Kit (ThermoFisher, Branchburg, NJ, USA) and purified using phenol-chloroform extraction and ethanolprecipitation. Adult female A. aegypti mosquitoes were injected with 500 ng dsRNA in PBS into the thorax to silence the A. aegypti CCT2 gene. At day 3 following dsRNA injection, the mosquitoes were injected with ZIKV Mex (100 PFU). At day 3, 7 days following virus injection, the whole mosquitoes were collected to examine the CCT2 expression levels by qRT-PCR to make sure the efficacy of dsRNA against CCT2 was as described below.

Quantitative Real-Time PCR
RNA from cells was extracted with RNeasy mini kit (Qiagen, Germantown, MD, USA), and the mosquito total RNA was extracted using TRIzol reagent (Ambion by Life Technologies, Branchburg, NJ, USA) according to manufacturer's protocol. DsRNA was produced using TranscriptAid T7 High Yield Transcription Kit (ThermoFisher, Branchburg, NJ, USA) and purified using phenol-chloroform extraction and isopropanol precipitation. The cDNA was generated with an iScript cDNA synthesis kit (Bio-Rad, Portland, ME, USA) according to manufacturer's protocol. Gene expression was examined by qRT-PCR using IQ SYBR Green Supermix. Viral RNA or Cct2 RNA levels were normalized to mosquito RP49 RNA levels according to 2 −∆∆Ct calculations.

Statistical Analysis
GraphPad Prism software (GraphPad, San Diego, CA, USA) was used to perform statistical analysis on all data. Mosquitoes were randomly allocated into different groups. No statistical methods were used to predetermine sample size. The viral titers were analyzed using two-way ANOVA with Bonferroni's multiple comparisons test or one-way ANOVA with Dunnett's multiple comparisons test. Rp49-normalized viral RNA levels were analyzed using the Wilcoxon-Mann-Whitney test. A p-value less than 0.05 was considered statistically significant, and all significant p-values are listed in the Figure legends.

Data Availability
Data that support the findings of this study are available from the corresponding authors upon request.

TRiC/CCT Complex Interacts with the Zika Virus NS1 Protein
To identify host proteins that interact with the Zika virus NS1 protein, we transfected 293T cells with a plasmid expressing Zika virus NS1 gene (corresponding to amino acid residues 1-352) with c-Myc tag at C-terminus. After removal of cellular debris, the supernatants from mock and NS1-transfected cells were incubated with anti-c-Myc magnetic beads then analyzed by SDS-PAGE, followed by silver staining (Figure 1a). Two unique regions of stained gel were subjected to in-gel trypsin digestion for protein identification, followed by LC-MS/MS. The unique identities of band 1 and band 2 are shown in Table 1. Interestingly, all subunits of the eight-subunit chaperonin containing TCP-1 (TRiC/CCT complex), whose theoretical masses of the identified proteins were compatible with their locations on the gel, were identified as binding partners to NS1 protein.    TRiC/CCT complex is a molecular chaperone belonging to the family of chaperonins, a conserved class of large double-ring complex of 800 kDa enclosing a central cavity, and is found in the cytoplasm of all eukaryotic cells [15,16]. It is composed of two identical rings, each composed of eight different subunits (CCT1-CCT8), and mediates cytosolic protein folding and assembly [17][18][19]. In order to examine the specificity of the interaction of Zika virus NS1 protein with subunits of TRiC/CCT complex, we cotransfected expression plasmids encoding HA-tagged CCT1-8 and nontagged NS1 protein, and performed coimmunoprecipitation assay. We found that CCT2, CCT4, CCT6, and CCT8 interact with NS1 protein (Figure 1b), suggesting NS1 protein uses the TRiC/CCT complex as a chaperone during ZIKV replication. TRiC/CCT complex is a molecular chaperone belonging to the family of chaperonins, a conserved class of large double-ring complex of 800 kDa enclosing a central cavity, and is found in the cytoplasm of all eukaryotic cells [15] [16]. It is composed of two identical rings, each composed of eight different subunits (CCT1-CCT8), and mediates cytosolic protein folding and assembly [17] [18] [19]. In order to examine the specificity of the interaction of Zika virus NS1 protein with subunits of TRiC/CCT complex, we cotransfected expression plasmids encoding HA-tagged CCT1-8 and nontagged NS1 protein, and performed coimmunoprecipitation assay. We found that CCT2, CCT4, CCT6, and CCT8 interact with NS1 protein (Figure 1b), suggesting NS1 protein uses the TRiC/CCT complex as a chaperone during ZIKV replication. Co-immunoprecipitation of NS1 with CCT1-8. HA-tagged CCT1-8 proteins and naïve (nontagged) NS1 protein were expressed in 293T cells. After immunoprecipitation with anti-HA beads, samples were analyzed by SDS-PAGE, followed by immunoblotting using a HA tag or NS1-specific antibody.

CCT2 Interacts with the Central Region of Zika Virus NS1
As CCT2 (T-complex protein 1 subunit beta) was confirmed to bind NS1 protein by immunoprecipitation, and the Mascot score, a statistical score for how well the experimental data match the database protein sequences, was highest among the eight subunits of TRiC/CCT complex, we further investigated the contribution of CCT2 to ZIKV replication. First, the colocalization of the ZIKV NS1 with CCT2 was examined by confocal microscopy (Figure 2a), suggesting that ZIKV NS1 and CCT2 were colocalized in the cytoplasm of HeLa cells. To determine the regions responsible for the interaction, we generated a series of NS1 deletion mutants with c-Myc-His tag at the C-termini of NS1 (Figure 2b), co-expressed them with CCT2, and immunoprecipitated with anti-c-Myc magnetic beads. The expression levels of NS1 deletion mutants and CCT2 were confirmed by immunoblotting with anti-c-Myc and anti-CCT2 antibodies, respectively (Figure 2c). Although the binding affinity differed in strength, NS1 deletion mutants NS1-CΔ52, NS1-CΔ112, NS1-NΔ60, and NS1-NΔ120 coimmunoprecipitated with CCT2. By contrast, NS1-NΔ180 rarely bound with CCT2 and two NS1

CCT2 Interacts with the Central Region of Zika Virus NS1
As CCT2 (T-complex protein 1 subunit beta) was confirmed to bind NS1 protein by immunoprecipitation, and the Mascot score, a statistical score for how well the experimental data match the database protein sequences, was highest among the eight subunits of TRiC/CCT complex, we further investigated the contribution of CCT2 to ZIKV replication. First, the colocalization of the ZIKV NS1 with CCT2 was examined by confocal microscopy (Figure 2a), suggesting that ZIKV NS1 and CCT2 were colocalized in the cytoplasm of HeLa cells. To determine the regions responsible for the interaction, we generated a series of NS1 deletion mutants with c-Myc-His tag at the C-termini of NS1 (Figure 2b), co-expressed them with CCT2, and immunoprecipitated with anti-c-Myc magnetic beads. The expression levels of NS1 deletion mutants and CCT2 were confirmed by immunoblotting with anti-c-Myc and anti-CCT2 antibodies, respectively (Figure 2c). Although the binding affinity differed in strength, NS1 deletion mutants NS1-C∆52, NS1-C∆112, NS1-N∆60, and NS1-N∆120 coimmunoprecipitated with CCT2. By contrast, NS1-N∆180 rarely bound with CCT2 and two NS1 deletion mutants (NS1-C∆172 and NS1-C∆232) failed to bind with CCT2. These data suggest that the region spanning amino acids at position 121-240 of NS1 is responsible for interaction with CCT2.

Interaction of ZIKV NS1 with CCT2 Depends on ATP Concentration
The central chamber of the TRiC/CCT complex folds many essential cellular proteins in an ATPdependent manner [11]. As we did not add any ATP to the buffer in our previous immunoprecipitation system, we examined the effect of ATP concentration on the interaction of NS1 with CCT2. Immunoprecipitation was performed with anti-c-Myc beads under several concentrations of ATP (0, 10, 50, 100 mM). We found that the interaction between CCT2 and NS1 was

Interaction of ZIKV NS1 with CCT2 Depends on ATP Concentration
The central chamber of the TRiC/CCT complex folds many essential cellular proteins in an ATP-dependent manner [11]. As we did not add any ATP to the buffer in our previous immunoprecipitation system, we examined the effect of ATP concentration on the interaction of NS1 with CCT2. Immunoprecipitation was performed with anti-c-Myc beads under several concentrations of ATP (0, 10, 50, 100 mM). We found that the interaction between CCT2 and NS1 was ATP-dependent (Figure 3), suggesting ATP concentration may modulate the binding between ZIKV NS1 and TRiC/CCT complex, contributing to the ZIKV replication.

Knockdown of CCT2 Reduces Zika Virus Replication in Mammalian Cells
To examine the biological function of CCT2 during the ZIKV life cycle, we generated shRNA-mediated knockdown (KD) of CCT2 in HeLa cells and assayed for virus replication. An shRNA targeting GFP was used as a negative control. At every 6 h after infection with ZIKV Cam , we harvested samples for testing protein expression and found that the expression levels of viral proteins and RNA were suppressed or delayed in CCT2-KD cells compared with control cells (Figure 4a,b). To investigate the effect of CCT2 knockdown on the production of infectious particles, we infected KD and control cells with ZIKV Cam . As shown in Figure 4c, downregulation of CCT2 expression significantly reduced the replication of ZIKV. These results suggest that TRiC/CCT complex supports ZIKV life cycle.
Viruses 2020, 12, x FOR PEER REVIEW 9 of 14 ATP-dependent (Figure 3), suggesting ATP concentration may modulate the binding between ZIKV NS1 and TRiC/CCT complex, contributing to the ZIKV replication.

Knockdown of CCT2 Reduces Zika Virus Replication in Mammalian Cells
To examine the biological function of CCT2 during the ZIKV life cycle, we generated shRNAmediated knockdown (KD) of CCT2 in HeLa cells and assayed for virus replication. An shRNA targeting GFP was used as a negative control. At every 6 h after infection with ZIKV Cam , we harvested samples for testing protein expression and found that the expression levels of viral proteins and RNA were suppressed or delayed in CCT2-KD cells compared with control cells (Figure 4a and b). To investigate the effect of CCT2 knockdown on the production of infectious particles, we infected KD and control cells with ZIKV Cam . As shown in Figure 4c, downregulation of CCT2 expression significantly reduced the replication of ZIKV. These results suggest that TRiC/CCT complex supports ZIKV life cycle.  (12,24, and 36 h), supernatants were collected, and virus titers were determined by plaque assay in Vero cells. **** p < 0.0001 by two-way ANOVA with multiple comparisons test. Data are plotted as the mean ± SEM.

Inhibition of TRiC/CCT Complex Function Using HSF1A Reduces Zika Virus Propagation
HSF1A is a cell-permeable compound that inhibits the function of TRiC/CCT [20]. To further elucidate the importance of TRiC/CCT complex during the ZIKV replication cycle, HeLa cells were treated with 0, 50, and 100 μM of HSF1A 12 h prior to ZIKV infection. The expression of viral proteins was suppressed in 50 and 100 μM HSF1A-treated cells compared with control cells with little toxicity (Figure 5a and 5b). The expression level of β-actin, whose expression/conformation is supported by TRiC/CCT complex, was not altered after HSF1A treatment, suggesting that the toxicity of HSF1A unlikely affects the expression level of viral proteins. To better understand the effect of HSF1A on the

Inhibition of TRiC/CCT Complex Function Using HSF1A Reduces Zika Virus Propagation
HSF1A is a cell-permeable compound that inhibits the function of TRiC/CCT [20]. To further elucidate the importance of TRiC/CCT complex during the ZIKV replication cycle, HeLa cells were treated with 0, 50, and 100 µM of HSF1A 12 h prior to ZIKV infection. The expression of viral proteins was suppressed in 50 and 100 µM HSF1A-treated cells compared with control cells with little toxicity (Figure 5a,b). The expression level of β-actin, whose expression/conformation is supported by TRiC/CCT complex, was not altered after HSF1A treatment, suggesting that the toxicity of HSF1A unlikely affects the expression level of viral proteins. To better understand the effect of HSF1A on the production of infectious particles, HeLa cells were treated with HSF1A 12 h prior to ZIKV infection. The amount of infectious virus in the supernatant was determined at 36 h post infection. Notably, ZIKV replication was significantly reduced in HeLa cells in a dose-dependent manner with little toxicity (Figure 5a,c). These findings support TRiC/CCT complex having an important role in the ZIKV replication cycle.
Viruses 2020, 12, x FOR PEER REVIEW 10 of 14 production of infectious particles, HeLa cells were treated with HSF1A 12 h prior to ZIKV infection. The amount of infectious virus in the supernatant was determined at 36 h post infection. Notably, ZIKV replication was significantly reduced in HeLa cells in a dose-dependent manner with little toxicity (Figure 5a and c). These findings support TRiC/CCT complex having an important role in the ZIKV replication cycle.

Suppression of CCT2 Reduces Zika Virus Burden in Mosquitoes
TRiC/CCT complex is conserved in all eukaryotic cells [21] [22] [23]. Thus, we hypothesized that this complex may regulate ZIKV infection in mosquitoes, which are vectors for ZIKV transmission cycle [24] [25] [26] [27]. In order to investigate the role of this complex in mosquitoes, we silenced the CCT2 gene in A. aegypti mosquitoes using RNAi, and then investigated whether suppression of CCT2 gene expression alters ZIKV replication in mosquitoes. After 3 days post dsRNA injection, A. aegypti mosquitoes were injected with 100 PFU of ZIKV Mex . Interestingly, there was a significant reduction in the levels of ZIKV in the CCT2 dsRNA-treated mosquitoes compared with the control GFP dsRNAtreated mosquitoes ( Figure 6). This result demonstrates that the TRiC/CCT complex contributes to ZIKV replication in mosquitoes as well as mammalian hosts.

Suppression of CCT2 Reduces Zika Virus Burden in Mosquitoes
TRiC/CCT complex is conserved in all eukaryotic cells [21][22][23]. Thus, we hypothesized that this complex may regulate ZIKV infection in mosquitoes, which are vectors for ZIKV transmission cycle [24][25][26][27]. In order to investigate the role of this complex in mosquitoes, we silenced the CCT2 gene in A. aegypti mosquitoes using RNAi, and then investigated whether suppression of CCT2 gene expression alters ZIKV replication in mosquitoes. After 3 days post dsRNA injection, A. aegypti mosquitoes were injected with 100 PFU of ZIKV Mex . Interestingly, there was a significant reduction in the levels of ZIKV in the CCT2 dsRNA-treated mosquitoes compared with the control GFP dsRNA-treated mosquitoes ( Figure 6). This result demonstrates that the TRiC/CCT complex contributes to ZIKV replication in mosquitoes as well as mammalian hosts.
CCT2 gene in A. aegypti mosquitoes using RNAi, and then investigated whether suppression of CCT2 gene expression alters ZIKV replication in mosquitoes. After 3 days post dsRNA injection, A. aegypti mosquitoes were injected with 100 PFU of ZIKV Mex . Interestingly, there was a significant reduction in the levels of ZIKV in the CCT2 dsRNA-treated mosquitoes compared with the control GFP dsRNAtreated mosquitoes ( Figure 6). This result demonstrates that the TRiC/CCT complex contributes to ZIKV replication in mosquitoes as well as mammalian hosts.

Discussion
Various screening methods for flavivirus-host protein interactions have been used to study the pathogenesis of flavivirus [12,28,29]. NS1 is a nonstructural protein highly conserved among the flaviviruses [30] and possesses multiple functions in the viral life cycle, including viral replication, immune evasion, and pathogenesis [8]. A previous study reported that an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 could induce Zika virus-specific antibody and T cell immune responses, thus providing protection against ZIKV challenge [9]. Also, mAbs targeting NS1 protein can protect against disease and death in a murine model [10]. These studies suggest that ZIKV NS1 could be a potential therapeutic target.
In this study, we identify the TRiC/CCT complex as a binding partner of the NS1 protein by pull-down assay followed by LC-MS/MS (Table 1). TRiC/CCT complex is a type II chaperonin composed of eight different subunits (CCT1-CCT8) that assists in the folding and assembly of essential host cytosolic proteins [31,32] and has been shown to be required for replication of several viruses [13,14,[33][34][35], including flaviviruses [12,36,37]. Here, we found that several subunits (CCT2, CCT4, CCT6A, and CCT8) of TRiC/CCT complex are able to bind ZIKV NS1 (Figure 1b), suggesting that NS1 protein uses the TRiC/CCT complex as a chaperone during ZIKV replication.
Our data demonstrated that the region spanning amino acids at position 121-240 of NS1 is responsible for interaction with CCT2 ( Figure 2). Several studies reporting the crystal structures of NS1 indicate that amino acids 121-240 might be involved in interactions with structural proteins Envelope (E) and precursor Membrane (prM) [38,39]. Interestingly, patients infected with Dengue virus (DENV) induce antibodies recognizing the region between amino acids 221 and 266 of DENV NS1, which is highly conserved among DENV serotypes [40]. Another study also demonstrated that the region located between amino acids 225 and 245 was highly conserved, which is beneficial for DENV vaccine design [41]. In addition, several monoclonal antibodies against WNV NS1 recognize amino acids 158 to 235, leading to strong protection against WNV in mice [42]. Considering the high structural similarity of NS1 among flaviviruses, targeting the 121-240 domain of the ZIKV NS1 could not only perturb NS1 function but also influence the interaction between the TRiC/CCT complex and NS1. Therefore, analyzing the spatiotemporal dynamics of NS1-CCT binding would be helpful to dissect this facet of host-pathogen interaction with greater precision.
Previous studies showed that the TRiC/CCT complex participates in the replication of several viruses, including DENV [12], hepatitis C virus [13], and reovirus [14]. Here, we found that the expression levels of viral proteins and RNA and the production of infectious particles were suppressed or delayed in CCT2-KD cells and in TRiC/CCT inhibitor-treated cells compared with control cells (Figures 4 and 5), suggesting that TRiC/CCT complex can be a therapeutic target against multiple viruses.
Interestingly, our result demonstrates that the TRiC/CCT complex contributes to ZIKV replication in mosquito vectors as well ( Figure 6). These data suggest the possible prevention of ZIKV spread in mosquitoes by using compounds targeting TRiC/CCT complex.
In conclusion, our study identified the TRiC/CCT complex as a binding partner of ZIKV NS1. Silencing the function of this complex reduces viral burden in mammalian cells and mosquitoes. These results highlight an important role for the relationship between NS1 and the TRiC/CCT complex during ZIKV infection. Although we need to investigate a possibility that viral RNA reduction in the CCT2 knockdown mosquitoes or viral reduction in the CCT2 knockdown cells is caused by diminished cell metabolism, we did not observe overt toxicity. Our results suggest that TRiC/CCT complex is exploited by ZIKV for maximal replication in both mammals and arthropod vectors and that TRIC/CCT complex may be a promising target for intervention.