PCV2 and PRV Coinfection Induces Endoplasmic Reticulum Stress via PERK-eIF2α-ATF4-CHOP and IRE1-XBP1-EDEM Pathways

Porcine circovirus 2 (PCV2) and pseudorabies virus (PRV) are two important pathogens in the pig industry. PCV2 or PRV infection can induce endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). However, the effect of PCV2 and PRV coinfection on the ERS and UPR pathways remains unclear. In this study, we found that PRV inhibited the proliferation of PCV2 mainly at 36 to 72 hpi, while PCV2 enhanced the proliferation of PRV in the middle stage of the infection. Notably, PRV is the main factor during coinfection. The results of the transcriptomic analysis showed that coinfection with PCV2 and PRV activated cellular ERS, and upregulated expressions of the ERS pathway-related proteins, including GRP78, eIF2α, and ATF4. Further research indicated that PRV played a dominant role in the sequential infection and coinfection of PCV2 and PRV. PCV2 and PRV coinfection induced the ERS activation via the PERK-eIF2α-ATF4-CHOP axis and IRE1-XBP1-EDEM pathway, and thus may enhance cell apoptosis and exacerbate the diseases.


Introduction
Porcine circovirus type 2 (PCV2) is one type of porcine circovirus, which belongs to the Circoviridae family Circovirus genus, containing a single-stranded, positive-sense, and circular DNA in the virion [1,2]. PCV2 is considered the causative pathogen of porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), and reproductive failure [1,2]. The virus infection has been reported in swine farms all over the world, with a high positive rate even exceeding 90%, and has caused great losses in the swine industry [1,2]. Notably, PCV2 infection causes subclinical symptoms, but it can also lead to immunosuppression, which provides a better condition for coinfection and/or the secondary infection of other pathogens [1,[3][4][5][6][7][8]. Furthermore, PCV2 infection can weaken the stimulation of the vaccine to the immune system and reduce the protective effect of the vaccine, or increase the virulence of the modified live vaccine [1,9]. Therefore, it is of great significance for the prevention and treatment of these clinical diseases to understand the situation and pathogenesis of concurrent infection.
Among the pathogens co-infected or sequentially infected with PCV2, viruses are the most important and should not be ignored, as they usually cause acute and serious diseases after co-or sequential infection [1,9,10]. As reported, the coinfection rates of PCV2 with porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (Suid herpesvirus 1 or PRV), classical swine fever virus (CSFV), and porcine epidemic diarrhea virus (PEDV) were 26.73%, 18.37%, 13.06%, and 3.47%, respectively, in Shandong province in China from 2015 to 2018 [11]. We recently evaluated samples isolated in 2018-2021 from Jilin province, China, and found that coinfection rates of PCV2 and PRV ranged from 1.19% to 11.93%.
PRV belongs to the genus Varicellovirus, Alphaherpesvirinae subfamily, and the family Herpesviridae. PRV infection can lead to nervous system disorder with high mortality in piglets, growth retardation and respiratory diseases in growing pigs, and reproductive failure in sows [11][12][13], and is one of the important diseases threatening the pig industry at present. It is worth noting that PRV can not only infect swine and wild boar but also can infect several mammals, including humans, dogs, and rodents [11,13,14].
In this study, porcine kidney epithelial cells (PK-15) infected with PCV2 and PRV alone, sequentially, or jointly for 12 h were analyzed by transcriptomic analysis, followed by evaluation of the differentially expressed genes (DEGs) and related ERS pathways involved in virus infection, which was helpful to further clarify the pathogenic mechanism of PCV2 and PRV coinfection.
The virulent wild-type PRV strain [23] and PCV2 strain CC1 [24] were used in this study.

Virus Infection
PK-15 cells (7 × 10 5 cells/well) were cultured in 12-well plates to reach 75% confluence. Then, cells were washed with phosphate buffer saline (PBS) three times and divided into 6 groups for virus infection.
Group 1 was used as a control group (PK-15 group). Group 2 was infected with PCV2 alone (PCV2 group), and group 3 was infected with PRV alone (PRV group). Cells were infected with PCV2 (multiplicity of infection, MOI = 0.5) or PRV (MOI = 0.01) alone for adsorption for 1 h. Then, cells were washed with PBS three times and then maintained in DMEM supplemented with 5% FBS for the indicated time, followed by evaluation via PCR, real-time PCR, or Western blotting. Group 4 and group 5 were two sequentially infected groups. In Group 4 (PCV2-12h-PRV group), cells were infected with PCV2 for 1 h, washed with PBS three times, and cultured in DMEM supplemented with 5% FBS. Then, 12 h later, cells were incubated with PRV for 1 h and washed with PBS three times, and cultured in DMEM supplemented with 5% FBS for the indicated time. In Group 5 (PRV-12h-PCV2 group), cells were infected with PRV for 1 h, washed with PBS three times and cultured in DMEM supplemented with 5% FBS. Then, 12 h later, cells were incubated with PCV2 for 1 h and washed with PBS three times and cultured in DMEM supplemented with 5% FBS for the indicated time. Then, samples were collected and evaluated via real-time PCR or Western blotting.
Group 6 (PCV2 + PRV group) was co-infected with PCV2 and PRV for 1 h. Then, cells were washed with PBS three times and cultured in DMEM supplemented with 5% FBS for the indicated time. Samples were collected and evaluated via PCR, real-time PCR, or Western blotting.

Real-Time Quantitative PCR (RT-qPCR)
Reverse transcription was conducted with the Fastking gDNA Dispelling RT SuperMix (Tiangen, Beijing, China) according to the manufacturer's instructions.
RT-qPCR was performed using 2×SYBR Green qPCR Master Mix (Bimake, Houston, TX, USA). The PCR program was as follows: 95 • C for 300 s, followed by 40 cycles of 95 • C for 15 s, 57 • C for 30 s, and 72 • C for 30 s, followed by melting curve analysis. Relative expression levels were calculated by using the 2 −∆∆ Ct method using GAPDH as the reference gene. The primer sequences are listed in Supplemental Table S1.

One-Step Growth Curve
The cells and supernatant of six groups were collected at 12-, 24-, 36-, 48-, 60-, and 72-h post-infection (hpi), followed by viral genomic DNA extraction using TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China). Viral DNA was determined by RT-qPCR and growth curves were generated based on the viral copies at the indicated time.

RNA-Seq Analysis
Total RNA was extracted by the TRNzol Universal Reagent (Tiangen, Beijing, China) from four groups mentioned above (PK-15, PCV2, PRV, and PCV2+PRV) at 12 hpi, and quantified using NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, Shanghai, China). Then, the total RNA was analyzed for transcriptome sequencing analysis by BGI Technology Co., Ltd. (Shenzhen, China). The library preparation and RNA-Seq were carried out by the DNBSEQ TM Sequencing platform.
Shanghai, China). After centrifugation at 12,000 rpm for 15 min at 4 • C, the supernatant was collected and quantified with a BCA protein assay kit (Beyotime, Shanghai, China).
The protein solution was mixed with 5× SDS-PAGE loading buffer and boiled for 10 min, followed by separation on 12% SDS-PAGE gel at 80 V for 3 h. Subsequently, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Merck, Darmstadt, Germany) at 300 mA for 60 min at 4 • C. The membranes were blocked in 5% skimmed milk (diluted with Tris-buffered saline containing 0.1% Tween 20, TBST) at room temperature for 90 min, and then incubated with the indicated primary antibodies dissolved in TBST overnight at 4 • C or room temperature for 2 h. After washing with TBST three times, the membrane was incubated with secondary antibodies HRP-conjugated goat anti-rabbit IgG (Wanleibio, Shenyang, China, 1:5000) or HRP-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China, 1:1000) at room temperature for 1 h. The signal was developed using the ECL reagent (Mei5bio, Beijing, China), and examined via Tanon 5200 system (Bioanalytical Imaging System, Azure Biosystems, Dublin, CA, USA). The mean densities of the protein bands were measured using ImageJ software. β-actin was used as an internal control.

Statistical Analysis
Two-way ANOVA statistical analysis was performed using GraphPad 7.0 (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Each value was the mean ± standard deviation (SD) of three replicates.

Growth Kinetics of PCV2 and PRV in Different Combinations of Infection Groups in PK-15 Cells
PK-15 cells were single-(PCV2 or PRV group), sequential-(groups PCV2-12h-PRV and PRV-12h-PCV2), or co-infected (PCV2 + PRV group) with PCV2 and/or PRV for 12, 24, 36, 48, 60, and 72 h, followed by evaluation of the virus replication in the cells. The results of growth curves showed that the copies of PCV2 were decreased in the sequential-and co-infected groups compared with that of the PCV2 infection alone ( Figure 1A), suggesting that PRV infection inhibits the proliferation of PCV2. The most obvious period of inhibition was at 36 to 72 hpi. Furthermore, the growth curves of Figure 1B showed that the copies of PRV were increased in the sequential-and co-infected groups compared with that of the PRV alone group at 24 to 36 hpi ( Figure 1B), suggesting that PCV2 infection enhances the proliferation of PRV, especially in the middle stage of the infection. In addition, the influence of the coinfection group on virus infection was similar to that of sequential infection groups.

The Influence of the Coinfection Group on Cell Viability Was Similar to That of Sequential Infection Groups
To evaluate the cell viability in the different infection groups, PK-15 cells were single-(PCV2 or PRV group), sequential-(groups PCV2-12h-PRV and PRV-12h-PCV2), or coinfected (PCV2+PRV group) with PCV2 and/or PRV for 12, 24, 36, 48, 60, and 72 h, followed by CCK8 assay. As shown in Figure 2A, cell viabilities were significantly decreased in one sequential infection group (PRV-12h-PCV2) and coinfection group compared with PCV2 infection alone at 36 to 60 hpi. However, there was no significant difference between the PCV2 alone group and sequential infection group PCV2-12h-PRV before 60 hpi. Moreover, the results of Figure 2B showed that sequential infection and coinfection have no significant difference in cell viabilities compared with PRV infection alone. These results indicate that PRV is the main factor for cell viability during the coinfection. In addition, the influence of the coinfection group on cell viability was similar to that of sequential infection groups. Therefore, we chose the coinfection group in the following study.

The Influence of the Coinfection Group on Cell Viability Was Similar to That of Sequential Infection Groups
To evaluate the cell viability in the different infection groups, PK-15 cells were single-(PCV2 or PRV group), sequential-(groups PCV2-12h-PRV and PRV-12h-PCV2), or coinfected (PCV2+PRV group) with PCV2 and/or PRV for 12, 24, 36, 48, 60, and 72 h, followed by CCK8 assay. As shown in Figure 2A, cell viabilities were significantly decreased in one sequential infection group (PRV-12h-PCV2) and coinfection group compared with PCV2 infection alone at 36 to 60 hpi. However, there was no significant difference between the PCV2 alone group and sequential infection group PCV2-12h-PRV before 60 hpi. Moreover, the results of Figure 2B showed that sequential infection and coinfection have no significant difference in cell viabilities compared with PRV infection alone. These results indicate that PRV is the main factor for cell viability during the coinfection. In addition, the influence of the coinfection group on cell viability was similar to that of sequential infection groups. Therefore, we chose the coinfection group in the following study.

The Influence of the Coinfection Group on Cell Viability Was Similar to That of Sequential Infection Groups
To evaluate the cell viability in the different infection groups, PK-15 cells were single-(PCV2 or PRV group), sequential-(groups PCV2-12h-PRV and PRV-12h-PCV2), or coinfected (PCV2+PRV group) with PCV2 and/or PRV for 12, 24, 36, 48, 60, and 72 h, followed by CCK8 assay. As shown in Figure 2A, cell viabilities were significantly decreased in one sequential infection group (PRV-12h-PCV2) and coinfection group compared with PCV2 infection alone at 36 to 60 hpi. However, there was no significant difference between the PCV2 alone group and sequential infection group PCV2-12h-PRV before 60 hpi. Moreover, the results of Figure 2B showed that sequential infection and coinfection have no significant difference in cell viabilities compared with PRV infection alone. These results indicate that PRV is the main factor for cell viability during the coinfection. In addition, the influence of the coinfection group on cell viability was similar to that of sequential infection groups. Therefore, we chose the coinfection group in the following study.

Transcriptomic Analysis Revealed Systemic Alterations of Gene Expressions in Cells Single-or Co-Infected with PCV2 and PRV
Transcriptomic RNA-seq analysis was performed on cells single-or co-infected with PCV2 and PRV for 12 h. Compared with the control group (PK-15), a total of 5143 differentially expressed genes (DEGs) were identified in PCV2-infected cells (2399 upregulated genes and 2744 downregulated genes), a total of 4122 DEGs were identified in PRV-infected cells (1970 upregulated genes and 2152 downregulated genes), and a total of 4825 DEGs were identified in PCV2 and PRV co-infected cells (2199 upregulated genes and 2626 downregulated genes) ( Figure 3A-C). Furthermore, a total of 3218 shared DEGs were identified in three infection groups via a Venn diagram ( Figure 3D).

Transcriptomic Analysis Revealed Systemic Alterations of Gene Expressions in Cells Singleor Co-Infected with PCV2 and PRV
Transcriptomic RNA-seq analysis was performed on cells single-or co-infected with PCV2 and PRV for 12 h. Compared with the control group (PK-15), a total of 5143 differentially expressed genes (DEGs) were identified in PCV2-infected cells (2399 upregulated genes and 2744 downregulated genes), a total of 4122 DEGs were identified in PRV-infected cells (1970 upregulated genes and 2152 downregulated genes), and a total of 4825 DEGs were identified in PCV2 and PRV co-infected cells (2199 upregulated genes and 2626 downregulated genes) ( Figure 3A-C). Furthermore, a total of 3218 shared DEGs were identified in three infection groups via a Venn diagram ( Figure 3D).  KEGG enrichment analysis of 3218 DEGs showed that the top 20 pathways included Ribosome biogenesis in eukaryotes, RNA transport, PI3K-Akt signaling pathway, MAPK signaling pathway, Hippo signaling pathway, Foxo signaling pathway, AMPK signaling pathway, HIF-1 signaling pathway, mTOR signaling pathway, Apoptosis, p53 signaling pathway, Wnt signaling pathway, Hippo signaling pathway-multiple species, ECMreceptor interaction, TGF-beta signaling pathway, TNF signaling pathway, ABC transporters, Protein processing in the endoplasmic reticulum (ER), Proteasome, and Protein export ( Figure 3E).
These results indicate that infection of PCV2 and PRV alone or simultaneously could activate ERS. Therefore, the influence of PCV2 and PRV coinfection on ER activation, especially the role of GRP78 (HSPA5), eIF2α, and ATF4 in the infection, will be verified in the follow-up research.

PCV2 and PRV Increased GRP78 Expression during Single-Infection and Coinfection
ER chaperones are important molecules involved in the ERS, among which two critical chaperone systems, calnexin/calreticulin, and GRP78/GRP94, have been widely studied for their roles in viral infection [25][26][27][28][29][30]. Therefore, we monitored the expression of GRP78 via real-time PCR and Western blotting. The result showed that the expression levels of GRP78 were upregulated in PCV2 single infected cells at 12 to 48 hpi, and were upregulated in PRV single infected at 4 to 12 hpi compared with that of the control group ( Figure 4A). The level of GRP78 was upregulated in PCV2 and PRV coinfection group at 4 and 12 hpi. These results were further confirmed by Western blotting, which showed that the levels of GRP78 were enhanced at 8 to 36 hpi in the virus single-infection and coinfection groups ( Figure 4B,C). These results suggest that PCV2 and PRV increased GRP78 expression during single-infection and coinfection, which may activate the ERS. among which HSPA5 was a key protein, which interacted with several proteins, including ERO1A (ERO1α), DNAJA2, HSPA8, HSPH1, eIF2α, and ATF4 ( Figure 3H). Moreover, biological network analysis of the 26 DEGs using KEGG assay showed that protein processing in the ER was associated with EIF2S1 (eIF2α), ATF4, and apoptosis pathway ( Figure 3I).
These results indicate that infection of PCV2 and PRV alone or simultaneously could activate ERS. Therefore, the influence of PCV2 and PRV coinfection on ER activation, especially the role of GRP78 (HSPA5), eIF2α, and ATF4 in the infection, will be verified in the follow-up research.

PCV2 and PRV Increased GRP78 Expression during Single-Infection and Coinfection
ER chaperones are important molecules involved in the ERS, among which two critical chaperone systems, calnexin/calreticulin, and GRP78/GRP94, have been widely studied for their roles in viral infection [25][26][27][28][29][30]. Therefore, we monitored the expression of GRP78 via real-time PCR and Western blotting. The result showed that the expression levels of GRP78 were upregulated in PCV2 single infected cells at 12 to 48 hpi, and were upregulated in PRV single infected at 4 to 12 hpi compared with that of the control group ( Figure 4A). The level of GRP78 was upregulated in PCV2 and PRV coinfection group at 4 and 12 hpi. These results were further confirmed by Western blotting, which showed that the levels of GRP78 were enhanced at 8 to 36 hpi in the virus single-infection and coinfection groups ( Figure 4B,C). These results suggest that PCV2 and PRV increased GRP78 expression during single-infection and coinfection, which may activate the ERS. . ****, p-value < 0.0001. βactin was used as a control. The relative amount of GRP78 to β-actin was quantified by densitometry analysis using ImageJ software (C). GRP78 (Affinity, Jiangsu, China, 1:2000), PRV-gD (Lvdu, Shandong, China, 1:500), PCV2-cap [31], and β-actin (Proteintech, Wuhan, Hubei, China, 1:10,000) were used as primary antibodies, respectively. Unprocessed original images can be found in Supplementary Figure S1.

PCV2 and PRV Infection Does Not Affect the Activation of the ATF6 Pathway
GRP78 can directly interact with three ERS sensors, ATF6, IRE1, and PERK, to regulate the UPR signaling pathways, and therefore activate ERS [25]. To clarify the downstream molecule of GRP78 during the PCV2 and PRV infection, expressions of ATF6

PCV2 and PRV Infection Does Not Affect the Activation of the ATF6 Pathway
GRP78 can directly interact with three ERS sensors, ATF6, IRE1, and PERK, to regulate the UPR signaling pathways, and therefore activate ERS [25]. To clarify the downstream molecule of GRP78 during the PCV2 and PRV infection, expressions of ATF6 were evaluated. The results showed that the copies of the atf6 gene were upregulated in the PCV2 + PRV co-infected group at 8 hpi ( Figure 5A), while no significant increase of ATF6 was observed in protein levels ( Figure 5B,C). These results indicate that PCV2 and PRV infection alone or coinfection does not activate the ATF6 pathway. were evaluated. The results showed that the copies of the atf6 gene were upregulated in the PCV2+PRV co-infected group at 8 hpi ( Figure 5A), while no significant increase of ATF6 was observed in protein levels ( Figure 5B,C). These results indicate that PCV2 and PRV infection alone or coinfection does not activate the ATF6 pathway.
To elucidate the role of the IRE1 pathway in PCV2 and/or PRV infection, expressions of IRE1α, EDEM1, as well as the splicing of xbp1 mRNA were examined. The results showed that phosphorylated IRE1α (p-IRE1) was increased in the PRV alone group from 4 to 24 hpi, and increased in the co-infected group at 24 hpi compared with that of the control group (PK-15) ( Figure 6A,B), suggesting that the IRE1 pathway was activated during PRV infection and PCV2+PRV coinfection. Similarly, the copies of xbp1 mRNA were upregulated at 8 hpi in both PRV alone and co-infected groups ( Figure 6C). Furthermore, the spliced xbp1 (sxbp1, 237 bp) and unspliced xbp1 (uxbp1, 263 bp) were detected in both PRV alone and co-infected groups from 12 to 48 hpi ( Figure 6D). Moreover, the copies of EDEM1 mRNA were upregulated in PRV alone group at 8 hpi, and a slight increase of EDEM1 mRNA can be detected in the co-infected group at 8 hpi ( Figure 6E). Levels of EDEM1 proteins were enhanced in the PRV group at 12 and 36 hpi, and in the co-infected group at 8 and 24 hpi ( Figure 6F). Notably, levels of p-IRE1/IRE1, EDEM1, and spliced xbp1 were not upregulated in the PCV2 single infected group. Therefore, these results suggest that the PRV infection activates the IRE1 pathway, while PCV2 has no obvious effect on the IRE1 pathway activation.
To elucidate the role of the IRE1 pathway in PCV2 and/or PRV infection, expressions of IRE1α, EDEM1, as well as the splicing of xbp1 mRNA were examined. The results showed that phosphorylated IRE1α (p-IRE1) was increased in the PRV alone group from 4 to 24 hpi, and increased in the co-infected group at 24 hpi compared with that of the control group (PK-15) ( Figure 6A,B), suggesting that the IRE1 pathway was activated during PRV infection and PCV2 + PRV coinfection. Similarly, the copies of xbp1 mRNA were upregulated at 8 hpi in both PRV alone and co-infected groups ( Figure 6C). Furthermore, the spliced xbp1 (sxbp1, 237 bp) and unspliced xbp1 (uxbp1, 263 bp) were detected in both PRV alone and co-infected groups from 12 to 48 hpi ( Figure 6D). Moreover, the copies of EDEM1 mRNA were upregulated in PRV alone group at 8 hpi, and a slight increase of EDEM1 mRNA can be detected in the co-infected group at 8 hpi ( Figure 6E). Levels of EDEM1 proteins were enhanced in the PRV group at 12 and 36 hpi, and in the co-infected group at 8 and 24 hpi ( Figure 6F). Notably, levels of p-IRE1/IRE1, EDEM1, and spliced xbp1 were not upregulated in the PCV2 single infected group. Therefore, these results suggest that the PRV infection activates the IRE1 pathway, while PCV2 has no obvious effect on the IRE1 pathway activation.

PCV2 and PRV Single-Infection and Coinfection Activated the UPR via the PERK-eIF2a-ATF4-CHOP Pathway
PERK belongs to the type I transmembrane protein of ER. Under ERS, PERK phosphorylates itself to form a dimer, and then phosphorylates eIF2α, which inhibits the translation of protein mRNA, reduces the entry of newly synthesized protein into ER, enhances the translation of ATF4, and induces the expression of pro-apoptosis protein C/EBP homologous protein (CHOP) [15,26,30]. It was reported that PCV2 infection selectively activates the PERK pathway via the PERK-eIF2α-ATF4-CHOP axis [16,33]. The replicase (Rep) and capsid (Cap) proteins of PCV2 can induce ERS by increasing the phosphorylation of PERK and then activating the eIF2α-ATF4-CHOP axis [33]. The viral Cap significantly decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage [33]. PCV2 can also reduce HMGB1 in the nuclei by regulating the PERK-ERO1α axis of ER, thus enhancing the viral replication [15]. Therefore, expressions of three downstream molecules of PERK, including eIF2α, ATF4, and CHOP, were evaluated. As shown in Figure 7A, copies of the ATF gene were enhanced in all the virus infection groups at 4 and 36 hpi compared with that of the control group (PK-15). Copies of the chop gene were enhanced in all the virus infection groups at 4 hpi compared with that of the control group ( Figure 7B). Furthermore, copies of the chop gene were also enhanced in the PCV2 single infection group at 8, 24, 36, and 48 hpi, which was consistent with the results reported

PCV2 and PRV Single-Infection and Coinfection Activated the UPR via the PERK-eIF2a-ATF4-CHOP Pathway
PERK belongs to the type I transmembrane protein of ER. Under ERS, PERK phosphorylates itself to form a dimer, and then phosphorylates eIF2α, which inhibits the translation of protein mRNA, reduces the entry of newly synthesized protein into ER, enhances the translation of ATF4, and induces the expression of pro-apoptosis protein C/EBP homologous protein (CHOP) [15,26,30]. It was reported that PCV2 infection selectively activates the PERK pathway via the PERK-eIF2α-ATF4-CHOP axis [16,33]. The replicase (Rep) and capsid (Cap) proteins of PCV2 can induce ERS by increasing the phosphorylation of PERK and then activating the eIF2α-ATF4-CHOP axis [33]. The viral Cap significantly decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage [33]. PCV2 can also reduce HMGB1 in the nuclei by regulating the PERK-ERO1α axis of ER, thus enhancing the viral replication [15]. Therefore, expressions of three downstream molecules of PERK, including eIF2α, ATF4, and CHOP, were evaluated. As shown in Figure 7A, copies of the ATF gene were enhanced in all the virus infection groups at 4 and 36 hpi compared with that of the control group (PK-15). Copies of the chop gene were enhanced in all the virus infection groups at 4 hpi compared with that of the control group ( Figure 7B). Furthermore, copies of the chop gene were also enhanced in the PCV2 single infection group at 8, 24, 36, and 48 hpi, which was consistent with the results reported previously [16]. To confirm these results, Western blotting was performed to examine p-eIF2α, eIF2α, ATF4, and CHOP. The results of Figure 7C,D demonstrated that levels of ATF4 were increased at 4-8 hpi and 24-48 hpi in the PCV2 single infection group, while increased at 4-8 hpi in PRV single infection group. The coinfection of PCV2 and PRV enhanced the ATF4 at 8, 24, and 36 hpi. Meanwhile, levels of CHOP proteins were increased at 4, and 36-48 hpi in the PCV2 single infection group, whereas the levels of CHOP were enhanced at 4 to 12 hpi in the PRV single-and co-infected groups ( Figure 7C,D). Moreover, ratios of p-eIF2α and eIF2α were significantly enhanced in all the infected groups at 4 to 48 hpi compared with the control group ( Figure 7C,E). Furthermore, PERK inhibitor GSK2656157 (Selleckchem, Houston, TX, USA) was used to further confirm the above result. As shown in Figure 8A, PERK inhibitor GSK2656157 has no obvious effect on cell viability. The virus infection was inhibited significantly by the inhibitor at 24, 36, 48, 60, and 72 hpi ( Figure 8B,C), suggesting that PCV2 and PRV infection was enhanced by the activation of the PERK pathway.
previously [16]. To confirm these results, Western blotting was performed to examine p-eIF2α, eIF2α, ATF4, and CHOP. The results of Figure 7C,D demonstrated that levels of ATF4 were increased at 4-8 hpi and 24-48 hpi in the PCV2 single infection group, while increased at 4-8 hpi in PRV single infection group. The coinfection of PCV2 and PRV enhanced the ATF4 at 8, 24, and 36 hpi. Meanwhile, levels of CHOP proteins were increased at 4, and 36-48 hpi in the PCV2 single infection group, whereas the levels of CHOP were enhanced at 4 to 12 hpi in the PRV single-and co-infected groups ( Figure 7C,D). Moreover, ratios of p-eIF2α and eIF2α were significantly enhanced in all the infected groups at 4 to 48 hpi compared with the control group ( Figure 7C,E). Furthermore, PERK inhibitor GSK2656157 (Selleckchem, Houston, TX, USA) was used to further confirm the above result. As shown in Figure 8A, PERK inhibitor GSK2656157 has no obvious effect on cell viability. The virus infection was inhibited significantly by the inhibitor at 24, 36, 48, 60, and 72 hpi ( Figure 8B,C), suggesting that PCV2 and PRV infection was enhanced by the activation of the PERK pathway.
These results indicate that the PERK-eIF2α-ATF4-CHOP axis was activated during PCV2 and PRV single-and coinfection, thus the UPR was promoted.

Discussion
In the present study, we found that PRV infection inhibits the proliferation of PCV2, and the most obvious period of inhibition was at 36 to 72 hpi ( Figure 1A). PCV2 infection enhances the proliferation of PRV, especially in the middle stage of the infection ( Figure  1B). During coinfection, PRV approached the peak of proliferation in 24 h, while the proliferation rate of PCV2 was slower than that of PRV ( Figure 1). PRV is the main factor for cell viability during the coinfection ( Figure 2).
As we all know, virus infection and replication need cellular replication and translation systems. Both PCV2 and PRV are virulent; however, PRV infection causes CPE, while PCV2 infection cannot. PCV2 is an extremely slow-growing virus, and the virus titer produced in cell culture is very low [34,35]. The life cycle of PRV is about 4-5 hpi (viral progeny can be examined within 4-5 hpi) [36], and that of PCV2 is about 24-36 hpi [34,35]. Therefore, PRV is more virulent than PCV2, thus inhibiting PCV2 infection due to CPE caused by PRV. Moreover, virion host shutoff (vhs) protein encoded by viral UL41 gene is a conserved protein in Alphaherpesviruses, which exhibits ribonuclease (RNase) activity and thus degrades mRNA, causing the shutdown of protein synthesis in host cells [36][37][38]. Meanwhile, the nuclease activity of Alphaherpesviruses vhs (HSV and PRV) can be stimulated by intracellular translation initiation factors, including eIF4A, eIF4B, and eIF4H [38][39][40], resulting in mRNA attenuation. Furthermore, the PRV UL41 protein preferentially targets the capped 3′-end of the internal ribosome entry site (IRES), but also cleaves downstream of the IRES region, suggesting the 5′ to 3′ RNase activity of PRV UL41 [38]. PCV2 induces immunosuppression, which is also beneficial for PRV infection. These are possible reasons why the copies of PCV2 decreased in the sequential-and co-infected groups compared with that of the PCV2 infection alone. These results further indicate that PRV plays a dominant role in the PCV2 and PRV coinfection. The exact mechanism of PRV inhibiting PCV2 remains to be studied, which is in progress in our lab. In addition, the influence of the coinfection group on virus infection was similar to that of sequential infection groups. Therefore, we chose the coinfection group (PCV2+PRV group) in the following study.
ER is an important organelle for virus infection and replication [27,30]. During infection, viral proteins are synthesized and processed in the ER, which may lead to the accumulation of unfolded and misfolded proteins in the ER lumen of the infected cells, and then induce ERS and UPR [27,30]. UPR consists of three signaling pathways, i.e., These results indicate that the PERK-eIF2α-ATF4-CHOP axis was activated during PCV2 and PRV single-and coinfection, thus the UPR was promoted.

Discussion
In the present study, we found that PRV infection inhibits the proliferation of PCV2, and the most obvious period of inhibition was at 36 to 72 hpi ( Figure 1A). PCV2 infection enhances the proliferation of PRV, especially in the middle stage of the infection ( Figure 1B). During coinfection, PRV approached the peak of proliferation in 24 h, while the proliferation rate of PCV2 was slower than that of PRV ( Figure 1). PRV is the main factor for cell viability during the coinfection ( Figure 2).
As we all know, virus infection and replication need cellular replication and translation systems. Both PCV2 and PRV are virulent; however, PRV infection causes CPE, while PCV2 infection cannot. PCV2 is an extremely slow-growing virus, and the virus titer produced in cell culture is very low [34,35]. The life cycle of PRV is about 4-5 hpi (viral progeny can be examined within 4-5 hpi) [36], and that of PCV2 is about 24-36 hpi [34,35]. Therefore, PRV is more virulent than PCV2, thus inhibiting PCV2 infection due to CPE caused by PRV. Moreover, virion host shutoff (vhs) protein encoded by viral UL41 gene is a conserved protein in Alphaherpesviruses, which exhibits ribonuclease (RNase) activity and thus degrades mRNA, causing the shutdown of protein synthesis in host cells [36][37][38]. Meanwhile, the nuclease activity of Alphaherpesviruses vhs (HSV and PRV) can be stimulated by intracellular translation initiation factors, including eIF4A, eIF4B, and eIF4H [38][39][40], resulting in mRNA attenuation. Furthermore, the PRV UL41 protein preferentially targets the capped 3 -end of the internal ribosome entry site (IRES), but also cleaves downstream of the IRES region, suggesting the 5 to 3 RNase activity of PRV UL41 [38]. PCV2 induces immunosuppression, which is also beneficial for PRV infection. These are possible reasons why the copies of PCV2 decreased in the sequential-and co-infected groups compared with that of the PCV2 infection alone. These results further indicate that PRV plays a dominant role in the PCV2 and PRV coinfection. The exact mechanism of PRV inhibiting PCV2 remains to be studied, which is in progress in our lab. In addition, the influence of the coinfection group on virus infection was similar to that of sequential infection groups. Therefore, we chose the coinfection group (PCV2+PRV group) in the following study.
ER is an important organelle for virus infection and replication [27,30]. During infection, viral proteins are synthesized and processed in the ER, which may lead to the accumulation of unfolded and misfolded proteins in the ER lumen of the infected cells, and then induce ERS and UPR [27,30]. UPR consists of three signaling pathways, i.e., PERK, IRE1α, and ATF6 pathways, that coordinate the adaptive response to ERS [27,30]. Furthermore, GRP78 can directly interact with three ERS sensors, ATF6, IRE1, and PERK, to regulate the UPR signaling pathways [25]. It was reported that PCV2 or PRV infection can induce ERS and UPR [15][16][17][18][19][20][21][22]. PCV2 plays a dominant role in the coinfection of PCV2 and CSFV [41]. However, there is no report on the ERS and UPR pathways caused by PCV2 and PRV coinfection. The results of the transcriptomic analysis showed that PCV2 and PRV coinfection activates ERS ( Figure 3D,E), and the ERS pathway-related proteins, GRP78, eIF2α, and ATF4, were enriched ( Figure 3F-H).
GRP78 is an ER chaperone, which can translocate from the ER to the plasma membrane, and act as an ERS sensor for virus infection, cancer, and other disorders in response to cellular stress [30,42]. Under normal conditions, GRP78 binds to transmembrane UPR sensors, IRE1, PERK, and ATF6, keeping them in inactive conformation [30,42,43]. During ER stress, GRP78 dissociates from the complex, which leads to the combination of unfolded or misfolded proteins and UPR sensors, and thus activates IRE1α, PERK, and/or ATF6 pathways [30,42]. We found in this study that PCV2 and PRV increased GRP78 expression during single-infection and coinfection (Figure 4). PRV alone infection and PCV2 + PRV coinfection activated the IRE1 pathway ( Figure 6), which is consistent with a previous report by Yang et al. [22], whereas the IRE pathway was not induced in the PCV2 single infected group (Figure 6), suggesting a dominant role of PRV in the activation of IRE1 pathway. Moreover, the PERK-eIF2α-ATF4-CHOP axis was activated during PCV2 and PRV single-and coinfection, thus the UPR was promoted (Figures 7 and 8).
It was reported that the PERK-ATF4-CHOP axis is one of the key pathways in ERSrelated apoptosis and autophagy [44,45]. CHOP is a necessary transcription factor for ERS-mediated cell death, and a critical signal to modulate the expression of Bcl-2 family members [46]. PCV2 infection selectively activates the PERK pathway via the PERK-eIF2α-ATF4-CHOP axis [16,33]. The replicase (Rep) and capsid (Cap) proteins of PCV2 can induce ERS by increasing the phosphorylation of PERK and then activating the eIF2α-ATF4-CHOP axis [33]. The viral Cap significantly decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage [33]. PCV2 also can reduce HMGB1 in the nuclei by regulating the PERK-ERO1α axis of ER, thus enhancing the viral replication [15]. Therefore, ERS can be induced during PCV2 and PRV single-and coinfections ( Figure 9). Infection with PCV2 mainly activates the PERK pathway, while infection with PRV can activate both PERK and IRE1 pathways. When PCV2 and PRV are co-infected, the PERK and IRE1 pathways can be activated simultaneously, whereas PRV plays a dominant role in the coinfection. We and other groups have found that coinfection of PCV2 and PRV ranged from 1.19% to 18.75% in the field [11,47], and is responsible for porcine respiratory disease complex (PRDC) and/or other diseases [1]. Therefore, PRV infection should be the primary target to prevent and treat the diseases caused by the coinfection of PCV2 and PRV. Figure 9. Schematic diagram of PCV2 and PRV coinfection on the activation of ERS. Arrow, confirmed; dashed arrow, needs to be clarified; ×, unactivated pathway.

Conclusions
In this study, we found that PRV played a dominant role in the sequential infection and coinfection of PCV2 and PRV. PCV2 and PRV coinfection induced the ERS activation via the PERK-eIF2α-ATF4-CHOP axis and IRE1-XBP1-EDEM pathway, thus enhancing cell apoptosis and exacerbating the diseases. Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.

Conclusions
In this study, we found that PRV played a dominant role in the sequential infection and coinfection of PCV2 and PRV. PCV2 and PRV coinfection induced the ERS activation via the PERK-eIF2α-ATF4-CHOP axis and IRE1-XBP1-EDEM pathway, thus enhancing cell apoptosis and exacerbating the diseases. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.