key: cord-1001621-1aei6s6h
authors: Guo, Kangkang; Zhang, Xiuping; Hou, Yufeng; Liu, Jing; Feng, Quanwen; Wang, Kai; Xu, Lei; Zhang, Yanming
title: A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response
date: 2020-10-17
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2020.108893
sha: c815d6c11f2010879ad039980a3949c172f33fdc
doc_id: 1001621
cord_uid: 1aei6s6h

Porcine circovirus type 2 (PCV2) infection causes porcine circovirus associated diseases (PCVAD) worldwide. Identification of host factors that interact with viral proteins is a fundamental step to understand the pathogenesis of PCV2. Our previous study reported that ORF5, a newly identified PCV2 viral protein supports PCV2 replication and interacts with multiple host factors. Here, we showed that a host factor YWHAB is an ORF5-interacting protein and play essential roles during PCV2 infection. By using protein-protein interaction assays, we confirmed that YWHAB directly interact with PCV2-ORF5 protein. We further showed that YWHAB expression was potently induced upon ORF5 overexpression and PCV2 infection. Remarkably, we found that the YWHAB strongly inhibited PCV2 replication, suggesting its role in defending PCV2 infection. By using the ectopic overexpression and gene knockdown approaches, we revealed that YWHAB inhibits PCV2-induced endoplasmic reticulum stress (ERS), autophagy, reactive oxygen species (ROS) production and apoptosis, suggesting its vital role in alleviating PCV2-induced cellular damage. Together, this study demonstrated that an ORF5-interacting host factor YWHAB that affects PCV2 infection and PCV2-induced cellular response, which expands the current understanding of YWHAB biological function and might serves as a new therapeutic target to manage PCV2 infection associated diseases.

Porcine circovirus type 2 (PCV2), with a 1.76 kb single-stranded circular DNA genome, is a member of the genus Circovirus in the family Circoviridae, causes porcine circovirus-associated diseases (PCVAD) worldwide (Lv et al., 2014; Meng, 2013) . As the smallest virus that can infect mammals, PCV2 genome contains 11 predicted open reading frames (ORFs), with 6 of them have been well-characterized .

Our previous study showed ORF4 suppresses PCV2-induced reactive oxygen species (ROS) accumulation in host cells (Lv et al., 2016) . The ORF5 was initially characterized as a novel viral protein that induces Endoplasmic Reticulum Stress (ERS) and activates NF-κB cascade (Lv et al., 2015) . By using ectopic overexpression approach, we further showed that the PCV2 ORF5 also induces Unfolded Protein Response (UPR) (Ouyang et al., 2019) . Our recent work demonstrated that PCV2 ORF5 induces autophagy in a AMPK-ERK1/2-mTOR signaling pathways dependent manner (Lv et al., 2020) . In addition, it is also reported that PCV2 ORF5 inhibits type I interferon (IFN) expression by inhibiting the genes required for IFN production and thus enhances PCV2 replication (Choi et al., 2018) . However, the underlying mechanism of how PCV2 ORF5 affects these host cellular responses remains enigmatic.

To investigate which host factors that interacts with PCV2 ORF5, yeast two-hybrid assay were performed and several host proteins including transmembrane glycoprotein NMB (GPNMB), cytochrome P450 1A1 (CYP1A1), 14-3-3β/α (YWHAB), zinc finger protein 511 isoform X2 (ZNF511) and serine/arginine-rich splicing factor 3 (SRSF3) were characterized (Lv et al., 2015) . By using transcriptome analysis, we found GPNMB expression was downregulated in PCV2-infected and PCV2 ORF5transfected cells, which suggests its important role in PCV2 infection (Guo et al., 2018) .

In particular, GPNMB interacts with PCV2 ORF5 and GPNMB inhibits PCV2 replication and ORF5 expression (Guo et al., 2018) . Mechanistically, GPNMB increases the Cyclin A expression and reduces S phase of host cells, which suppresses PCV2 replication (Guo et al., 2018) . That study validated the result obtained from two-hybrid assay and showed that GPNMB restricts PCV2 replication. However, the interaction between J o u r n a l P r e -p r o o f PCV2 ORF5 and other characterized host factors were still unknown.

Among these ORF5-interacting host factors, 14-3-3β/α (also known as YWHAB, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) piqued our interest for its role in viral infection . The 14-3-3β/α protein belongs to the highly conserved 14-3-3 protein family that widely distributed from plants to mammals (Obsilova et al., 2008) . The 14-3-3 family consists of seven isoforms (β, ε, η, γ, θ, ζ and σ), which were encoded by seven distinct genes (YWHAB, YWAHE, YWHAH, YWHAG, YWHAQ, YWHAZ, and SFN) (Rosenquist et al., 2000) . The 14-3-3 family members are involved in various cellular processes including signal transduction, cell cycle, cell proliferation, apoptosis, differentiation and survival (Fu et al., 2000; Mackintosh, 2004; van Hemert et al., 2001; Xing et al., 2000) . The target of 14-3-3 in cells are very diverse, with more than 300 different proteins have been identified and these protein were involved in diverse essential cellular processes including cell-cycle regulation, apoptosis, metabolism, protein trafficking and signal transduction (Morrison, 2009) . Accumulating evidence suggests that the isoforms of 14-3-3 proteins, in particular YWHAB, affects virus infection by different mechanisms Hwang et al., 2018; Nathan and Lal, 2020; Toshima et al., 2001) .

Thus, 14-3-3 family proteins may represent new diagnostic and therapeutic targets for virus infections. However, the interaction between PCV2 ORF5 protein and 14-3-3 family proteins and their role of 14-3-3 family proteins in regulating PCV2 infection remains unclear.

In this study, based on our previous finding that 14-3-3β/α (YWHAB) interacts with ORF5, we aimed to delineate the relationship between YWHAB and ORF5 protein during PCV2 infection. The ectopic gene expression or knockdown approaches were employed to overexpress or silence YWHAB. We found that YWHAB specifically binds to PCV2 ORF5 while PCV2 infection and ORF5 overexpression induces the expression of YWHAB. Remarkably, we showed that YWHAB inhibits PCV2-induced Endoplasmic Reticulum Stress (ERS), autophagy, ROS production and apoptosis, J o u r n a l P r e -p r o o f which suggesting its essential roles in alleviating PCV2 infection induced cellular damages. Together, this study revealed that a host factor YWHAB interacts with PCV2 ORF5 protein and inhibits PCV2 infection, which provides new insight into the pathogenic mechanisms of PCV2 and represent a novel therapeutic target in constraining PCV2 infection.

Porcine kidney PK-15 cells (ATCC: CCL-33) and human embryonic kidney HEK293 (ATCC: CRL-1573) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Solarbio, China), supplemented with 10% fetal bovine serum (FBS) (Gibco, United Kingdom) and penicillin (100 U/mL) and streptomycin (100 μg/mL). Porcine alveolar macrophages 3D4/2 (PAMs) (ATCC: CRL-2845) were grown in RPMI 1640 medium (Solarbio, China) with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL).

Delivery of eukaryotic expression plasmids into cells was performed by using the TurboFect Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

The YWHAB gene (14-3-3β/α, GenBank No. XP-005673018.1) was amplified from the cDNA of PK-15 cells by using the reverse transcription-PCR (RT-PCR) with primer pairs listed in Table S1 and then subcloned into the pCDH-CMV-MCS-EF1 vector with a Flag tag to generate CMV-Flag-YWHAB. The PCR product was also inserted into the pDsRed-N and pGEX-6P-1 vectors to generate pDsRed-YWHAB-N1 and pGEX-6P-1-YWHAB, respectively. Three pairs of shRNAs targeting to YWHAB gene and a random sequence negative control naming shN were predicted and designed (http://rnaidesigner.thermofisher.com). The fragments were cloned into pCDH-U6-MCS-EF1GreenPuro vector after annealing to generate shYWHAB and shN lentivectors. The integrity of all plasmids was confirmed by sequencing. All primers were listed in Table S1 .

The wild-type PCV2 Yangling strain (wPCV2) was kept in our laboratory (Tang et al., 2011) . PK-15 or PAMs cells were infected with PCV2 with at indicated multiplicity of infection (MOI) for 1 h, then replaced with fresh RPMI 1640 medium with 2% FBS.

Total cell RNA was isolated using TRIzol Reagent (Takara, Japan), then reverse transcribed into cDNA using Fasking RT kit (Takara, Japan) according to the manufacturers' instructions. To determine the expression level of targeted gene, quantitative real-time PCR was conducted using SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa). The primers were designed and synthesized by AUGCT Biotech sequences (Beijing, China) (Table S1 ). Finally, the relative mRNA expression level of each gene were assessed using the 2 -∆∆Ct method and normalized to the housekeeping gene β-actin. A non-treatment (Mock) PK-15 cells served as the negative control. 

The cells were treated with trypsin without EDTA and resuspended to final concentration of 1 × 10 5 cells/mL. They were stained with 5 μL Annexin V-FITC and 5 μL 7-aminoactinomycin D (7-AAD) for 10 min in the dark at room temperature or the cells were incubated with 2',7'-dichlorofluorescein diacetate (DHE, 5 μM, Beyotime, China) at 37 °C for 30 min. The apoptotic cell populations and the ROS production level were determined by fluorescence microscopy and flow cytometry. Fluorescence intensity was collected at 518 nm and 605 nm under the logarithmic mode. The data were analyzed using FlowJo_v10 software.

HEK293 cells were co-transfected with pEGFP-ORF5 and CMV-Flag-YWHAB plasmids and harvested at 48 h post-transfection by using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing PMSF for 30 min at 4°C. Cell lysate was collected by centrifuging for 30 min at 12 000 rpm and then incubated with anti-Flag A+G-agarose beads (Sigma-Aldrich, United States) at 4°C overnight. The beads were collected by centrifugation and washed 3 times with ice-cold TBST.

Immunoprecipitated proteins were extracted from the agarose beads by boiling for 10 min in 5× loading buffer and then subjected to SDS-PAGE and incubated with anti-GFP antibody as describe above.

The GST-YWHAB protein was produced from Escherichia coli BL21 and treated with pull-down lysis buffer (Thermo Fisher Scientific, United States) and then conjugated to J o u r n a l P r e -p r o o f glutathione agarose beads for 2 h at 4 °C. Next, the beads were washed with 1:1 wash solution (TBS: pull-down lysis buffer) for five times and incubated with pEGFP-ORF5 harvested from HEK293 cells overnight at 4°C. After washed for five times, the bound proteins were analyzed by Immunoblotting.

PAMs cells were co-transfected with pEGFP-ORF5 and pDsRed-YWHAB plasmids by TurboFect Transfection Reagent (Thermo Fisher Scientific). As a negative control, pEGFP-C1 and pDsRed-N1 plasmids were subjected to same experimental conditions.

At 36 h post-infection, the cultured cells were washed with PBS and then fixed with 4% paraformaldehyde diluted in PBS. Cell nuclei were stained with 4',6'-diaminido-2phenylindole (DAPI) for 10 min at room temperature. The cells were imaged under laser confocal scanning microscopy (Carl Zeiss, Germany).

All data were presented as mean ± SD. Comparisons between groups were determined with the Student's t-test. One-way analysis of variance (ANOVA) difference test was used to compare the data from pairs of treated or untreated groups. Statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

In our previous study, by using the yeast two-hybrid assay, several proteins (YWHAB, GPNMB, CYP1A1, ZNF511 and SRSF3) were identified as ORF5-interacting host factors. To further validate the interaction between YWHAB (also known as 14-3-3β/α) and ORF5 protein, the CMV-Flag-YWHAB and pEGFP-ORF5 plasmids were cotransfected into HEK293 cells. The result showed a clear binding between YWHAB and PCV2 ORF5 ( Figure 1A ), suggesting the potential interaction between ORF5 and YWHAB. Next, the GST pull-down assay also confirmed a specific interaction between YWHAB and PCV2 ORF5 protein ( Figure 1B) , which purified GST-YWHAB protein and cell lysis from ORF5-overexpressed cells were employed. Furthermore, colocalization J o u r n a l P r e -p r o o f of YWHAB and PCV2 ORF5 protein was observed in the cytoplasm of pEGFP-ORF5 and pDsRed-YWHAB vectors co-transfected porcine alveolar macrophages 3D4/2 (PAMs) cells ( Figure 1C) , which further fostered an interaction between ORF5 and YWHAB. Together, these results confirmed that PCV2 ORF5 interacted with the host factor YWHAB.

We have already shown that PCV2 ORF5 interact with YWHAB. However, whether PCV2 infection or ORF5 transfection could affect the expression of YWHAB remains unknown. To this aim, the endogenous YWHAB levels were measured upon PCV2 infection and ORF5 transfection. We found that PCV2 infection substantially induces YWHAB expression at mRNA and protein level from 24 h post-infection ( Figure 2A, 2B and 2C). Correspondingly, the expression of YWHAB was induced to a higher level with the increase of infection MOI ( Figure 2D , 2E and 2F). The interaction between PCV2 ORF5 and host factor YWHAB prompts us to test whether the PCV2 ORF5 protein could affect YWHAB expression. As expected, the mRNA expression of YWHAB was significantly induced by ORF5 transfection ( Figure 2G ). Consistently, the protein expression of YWHAB was upregulated by PCV2 ORF5 (Figure 2H and 2I).

Together, these results demonstrated that PCV2 infection and ORF5 transfection induced YWHAB expression at both transcriptional and translational levels.

Although have been shown that YWHAB expression was greatly induced by PCV2 infection, the role of YWHAB during PCV2 infection is still enigmatic. To investigate the potential function of YWHAB in affecting PCV2 infection, the lentiviral-based overexpression and knockdown approaches were employed. As shown in Figure 3A As shown in Figure 3F , a successful knockdown of YWHAB were obtained and following studies were performed on the shYWHAB-3 cell lines ( Figure 3F ). Conversely, the PCV2 replication level was significantly increased in YWHAB-knockdown cells ( Figure 3G ). The Cap protein expression and ORF5 gene expressions were also upregulated in YWHAB silenced cells ( Figure 3H, 3I and 3J ). Together, these results convincingly demonstrated that YWHAB inhibits PCV2 replication.

PCV2 infection and ORF5 protein induces the Endoplasmic Reticulum Stress (ERS) (Lv et al., 2015) . To investigate whether the YWHAB protein could affect the PCV2induced ERS, we measured the expression of GRP78 and GRP94, which are the hall markers of ERS, in YWHAB overexpressed or silenced cells upon PCV2 infection. Both mRNA expressions of GRP78 and GRP94 were significantly decreased in YWHAB overexpressed cells at 24 and 48 h post PCV2 infection ( Figure 4A and 4B) .

Consistently, the protein expression levels of GRP78 and GRP94 was also induced to a lower level by PCV2 in YWHAB overexpressed cells ( Figure 4C, 4D and 4E ). In contrast, the gene expression of GRP78 and GRP94 was elevated in YWHAB silenced cells at 24 h and 48 h post PCV2 infection ( Figure 5A and 5B). Importantly, similar results were obtained for the protein expression level of GRP78 and GRP94 in YWHAB silenced cells ( Figure 5C , 5D and 5E). Together, we revealed that YWHAB alleviated the PCV2-induced endoplasmic reticulum stress.

Our previous work reported that ORF5 protein induces autophagy through PERK-eIF2a-ATF4 and AMPK-ERK1/2-mTOR pathways to promotes viral replication (Lv et al., 2020) . To determine whether YWHAB affect the PCV2-induced autophagy, the autophagy markers Beclin1 and LC3-II were measured in YWHAB overexpressed or silenced cells. Importantly, PCV2 infection induced lower level of Beclin-1 gene expression in YWHAB overexpressed cells compared to that in control cells ( Figure   J o u r n a l P r e -p r o o f 6A). In addition, the protein expression of LC3-II, the autophagy marker, was also less activated in YWHAB overexpressed cells ( Figure 6B and 6C ). In contrast, PCV2 infection induced stronger gene expression of Beclin-1 in YWHAB knockdown cells ( Figure 6D ) and similar result was obtained for the protein expression of LC3-II ( Figure   6E and 6F). Together, these results demonstrated that YWHAB inhibits the PCV2induced autophagy.

The intracellular ROS production is an important indicator of the cellular stresses.

Previous studies showed that PCV2 infection induces the production of intracellular ROS Zhang et al., 2019) . To investigate the role of YWHAB in regulating PCV2-induced ROS production, we measured the intracellular ROS in YWHAB overexpressed and knockdown cells by using flow cytometry. In line with a previous study , we confirmed that PCV2 infection strongly induced intracellular ROS level. Importantly, the intracellular ROS was significantly decreased in YWHAB upregulated cells (Figure 7) . Conversely, the intracellular ROS level was significantly elevated in the cells that have lower YWHAB expression (Figure 7) . This result indicated that YWHAB inhibits PCV2-induced ROS.

It has been demonstrated that PCV2 infection induces apoptosis in both cell culture model and animal model (Chang et al., 2007; Resendes et al., 2011) . To explore whether the YWHAB affects the PCV2-induced apoptosis, the apoptosis in PCV2infected YWHAB overexpressed or knockdown cells was measured at 24 h postinfection. As shown in Figure 8A , the PCV2-induced apoptosis was strongly attenuated in YWHAB overexpressed cells compared to that in Lv control cells. As expected, the PCV2 infection activated apoptosis was potentiated in YWHAB silenced cells ( Figure   8B ). Together, this result suggested that YWHAB inhibits cells from PCV2-induced apoptosis.

The identification of host factors that interact with viral proteins is essential to understand pathogenesis of virus. In our previous study, five host proteins (YWHAB, GPNMB, CYP1A1, ZNF511 and SRSF3) were identified as PCV2 ORF5-interacting factors by used yeast two-hybrid assay (Lv et al., 2015) . The interaction between GPNMB and PCV2 ORF5 were confirmed through different approaches and we surprisingly found that GPNMB inhibits PCV2 replication and ORF5 expression by regulating the Cyclin A expression in host cells (Guo et al., 2018) . This study validated our previous screening approach and prompt us to investigate the role of other ORF5interacting proteins in PCV2 infection. Intrigued by its role in regulating diverse cellular processes and affecting virus replication, we focused on interaction between YWHAB and PCV2 in present study. A specifical interaction between PCV2-ORF5 and YWHAB was validated via diffident approaches (Figure 1) . Then, we surprisingly found that PCV2 infection and ORF5 transfection strongly activated YWHAB expression. This is consistent with a previous study that showed pancreatic necrosis virus (IPNV) induced the expression of a large number of genes including YWHAB (14-3-3β), but they did not illustrate the role of YWHAB against IPNV infection (Villalba et al., 2017) . In this study, we further showed that YWHAB inhibits PCV2 replication. Several isoforms of 14-3-3 proteins has been reported affect viral infection (Aoki et al., 2000; Diao et al., 2001; Kino et al., 2005) . In particular, a study shows that the M protein of parainfluenza virus 5 (PIV5) interacts with host protein 14-3-3 β and the production of PIV5 particles was negatively affected (Schmitt and Lamb, 2004) . Together, we believe that YWHAB is an anti-PCV2 host factor, in which the expression is induced upon viral infection.

However, the molecular mechanism of how the YWHAB is induced needs further investigation.

The PCV2 ORF5 protein has been evidenced to induce the Endoplasmic Reticulum Stress (ERS) and unfolded protein response (UPR) (Ouyang et al., 2019) . In present study, we found that a host factor YWHAB alleviates PCV2-induced ERS. The hallmark J o u r n a l P r e -p r o o f of ERS is the upregulation of glucose-regulated protein 78 (GRP78) and 94 (GRP94) (Bailey and O'Hare, 2007) . During ERS, BiP/GRP78 or GRP94 binds to misfolded proteins and releases PERK, ATF6, and IRE1, resulting in their activation and initiation of the UPR (Tu and Weissman, 2004) . In this study, we found that YWHAB significantly inhibits GRP78 and GRP94 expression upon PCV2 infection (Figure 3 and 4) . PCV2

infection triggers autophagy to facilitate its replication (Zhai et al., 2019) . Our previous work showed that ORF5 protein induces autophagy through PERK-eIF2a-ATF4 and AMPK-ERK1/2-mTOR pathways to promotes viral replication (Lv et al., 2020) . Given the importance of autophagy in regulating virus replication, it is not surprising that both PCV2 infection and host factors YWHAB can alter autophagy. Although no study has been reported that PCV2 ORF5 protein induces ROS production, PCV2 infection increases ROS production to facilitate PCV2 replication (Chen et al., 2012) . PCV2 infection induced ROS production elicits dynamic relative protein1 (Drp1) phosphorylation and activation of the PINK1/Parkin pathway, which eventually activates mitophagy and mitochondrial apoptosis . In this study, PCV2 infection induced ROS production is also observed ( Figure 6 ) and YWHAB potently decreases this effect. However, whether YWHAB inhibits PCV2 infection by suppressing GRP78/GRP94, autophagy or ROS production needs further investigation.

Together, these results showed that the ORF5-interacting protein YWHAB potently alleviates the ORF5-elicited ERS, autophagy and ROS production. Nevertheless, further study is required to elucidate the underlying molecular mechanism of YWHAB in mitigating these cellular responses.

The 14-3-3 family proteins participates in versatile cell cascades through its phosphoserine and phosphothreonine binding activity (Berg et al., 2003; Weinert et al., 2019) . In particular, recent studies revealed that 14-3-3 family proteins are involved in viral infection with diverse mechanisms Hwang et al., 2018; Nathan and Lal, 2020; Toshima et al., 2001) . The YWHAB protein interacting with glycoprotein of spring viremia of carp virus positively affects viral attachment and entry . Some 14-3-3 family protein directly act on the viral protein like the severe acute J o u r n a l P r e -p r o o f respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein gets serinephosphorylated and translocated to the cytoplasm by binding to 14-3-3 proteins (Surjit et al., 2005) . On the other hand, some 14-3-3 proteins alter the cellular responses to affect virus infection. For instance, 14-3-3ε stabilizes the interaction between RIG-I with TRIM25 to facilitate RIG-I ubiquitination, which confers innate immune response against hepatitis C virus and other pathogenic RNA viruses (Liu et al., 2012) .

Furthermore, binding of dengue virus NS3 protein to 14-3-3ε prevent the translocation of RIG-I to adaptor protein MAVS, thus blocking the antiviral signaling (Chan and Gack, 2016) . Herein, we showed that interaction of the YWHAB protein potently decreased the PCV2-induced cellular damages that could negatively affect PCV2 replication.

In summary, in this study we revealed that a host factor YWHAB interacts with PCV2- Data in (C) is one representative of those from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. 

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