Regulation of Apoptosis During Porcine Circovirus Type 2 Infection

Apoptosis, an indispensable innate immune mechanism, regulates cellular homeostasis by removing unnecessary or damaged cells. It contains three signaling pathways: the mitochondria-mediated pathway, the death receptor pathway and the endoplasmic reticulum pathway. The importance of apoptosis in host defenses is stressed by the observation that multiple viruses have evolved various strategies to inhibit apoptosis, thereby blunting the host immune responses and promoting viral propagation. Porcine Circovirus type 2 (PCV2) utilizes various strategies to induce or inhibit programmed cell death. In this article, we review the latest research progress of the apoptosis mechanisms during infection with PCV2, including several proteins of PCV2 regulate apoptosis via interacting with host proteins and multiple signaling pathways involved in PCV2-induced apoptosis, which provides scientific basis for the pathogenesis and prevention of PCV2.

Porcine circovirus has 11 potential ORFs, so far to date, four of them have been characterized as functional proteins in replicating PCV2, including ORF1 to ORF4 (Hamel et al., 1998;Lv et al., 2014a;Hong et al., 2015), while only three ORFs have been recognized for PCV1 and PCV3: ORF1 to ORF3 (Saraiva et al., 2018). The ORF1 encodes two replicases (Rep and Rep'), the Rep proteins of PCV-1 and PCV-2 are similar in size and are responsible for the replication of the circoviral genome (Mankertz, 2012). The capsid protein encoded by ORF2 is the sole structural protein of PCV2 and contains a highly conserved basic amino acid sequence (Timmusk et al., 2008;Latini et al., 2011); therefore, it contains the major antigenic determinants of the virus (Nawagitgul et al., 2000). However, the three proteins of PCV3 are less similar to those of PCV1 and PCV2 (Palinski et al., 2016;Phan et al., 2016). The proteins encoded by ORF3 and ORF4 genes are not required for viral replication, but are closely related to the spread and virulence of the virus (Lv et al., 2015a). The protein encoded by ORF3 gene plays a vital role in the pathogenesis of the virus through its apoptotic activity in vitro and in vivo (Liu et al., 2005Lin et al., 2011). The ORF4 protein is capable of blocking PCV2-induced apoptosis by bringing down caspases activities (Gao et al., 2014a;Lv et al., 2015b). Besides this, a novel ORF5 protein has recently been discovered in PCV2-infected cells and may be involve in activation of NF-κB pathway (Lv et al., 2015a).
Apoptosis, also called programmed cell death, is an indispensable defense mechanism for host resistance to pathogens invasion (Jorgensen et al., 2017). Apoptosis is strictly regulated and can be triggered by multiple stimuli such as normal development, pathogen infection and several factors leading to disruption of cellular functions (Tait and Green, 2010;Czabotar et al., 2014). Apoptotic cells exhibit characteristic morphological abnormalities including chromatin condensation, nuclear fragmentation, membrane blebbing, and apoptotic body formation (Kroemer et al., 2005;Galluzzi et al., 2007). Apoptosis classically occurs via the intrinsic pathway (also called the mitochondrial pathway), the extrinsic pathway (also called the death receptor pathway) and the ER pathway (Hong et al., 2015). In brief, the mitochondrial pathway is induced by a variety of intracellular signals, such as hypoxia, nutrient deprivation and oxidative stress, which cause MOMP . Subsequently, AIF, cyt c and Smac/DIABLO are released from the mitochondrial membrane to the cytoplasm Galluzzi et al., 2012). Cyt c can recruit pro-caspase9 and apoptotic protease activating factor-1 (apaf-1) to form an apoptosome, which subsequently activates downstream executioner caspases to trigger apoptosis (Li et al., 1997;Acehan et al., 2002). In addition, the mitochondrial pathway is mainly regulated by Bcl-2 (B-cell lymphoma 2) family proteins, which are classified into three types (Cory and Adams, 2002). One is an anti-apoptotic sub-family, which includes Bcl-xL (B-cell lymphoma-extra large) and Bcl-2. Another is pro-apoptotic BH3 only proteins, such as Bid (BH3 interacting-domain death agonist) and Bad (Bcl-2 associated death promoter), these proteins are antagonists to the anti-apoptotic sub-family proteins. The third sub-family includes Bak (Bcl-2 homologous antagonist killer) and Bax (Bcl-2 associated x protein). On the other hand, the death receptor pathway is activated by the binding of a specific ligand to the corresponding death receptor, resulting in activation of caspase8 and caspase3, which finally leads to cleavage of cellular DNA (Lamkanfi et al., 2007;Tummers and Green, 2017). What's more, ER stress regulates the concentration of Ca 2+ and initiates the IRE1, PERK, and ATF6 pathways, which are associated with the mitochondrial pathway of apoptosis (Shore et al., 2011;Verma and Datta, 2012).
Porcine Circovirus type 2 infection induces apoptosis both in vitro and in vivo (Chang et al., 2007a;Seeliger et al., 2007;Galindocardiel et al., 2011;Resendes et al., 2011;Sinha et al., 2012), it has been reported that PCV2 can induce B lymphocyte deletion through apoptosis and macrophage apoptosis can be detected in the spleen of PCV2 infected mice (Shibahara et al., 2000), so apoptotic cell death may be one of the causes of lymphopenia after PCV2 infection (Resendes et al., 2004). Similarly, apoptosis is one of the causes of lymph node loss and hepatocyte decline in pigs with PMWS (Krakowka et al., 2004). Apoptosis has also been proposed as a natural part of the viral life cycle (Young et al., 2007), in the early stage of PCV2 infection, PCV2 may prevent apoptosis by expressing its anti-apoptotic gene to accomplish its propagation, while apoptosis may be a powerful strategy for the release and dissemination of progeny virions in the late stage (Liu et al., 2005). However, the molecular mechanism of PCV2-regulated apoptosis is still unclear. In this article, we focus on reviewing the roles of PCV2 in the process of apoptosis, which is useful for future research.  (Lv et al., 2014 pathogenesis mechanisms of PCV2, the interactions between the PCV2 Cap protein with nine different cellular proteins were confirmed (Table 1), including complement factor C1qB, the receptor protein for the gC1qR, MKRN1, cell adhesion molecule P-selectin, prostate apoptosis response-4 (Par-4) protein, NAP1, NPM1, Hsp70 and Hsp40 (Timmusk et al., 2006a;Finsterbusch et al., 2009b;Liu et al., 2013b). However, only MKRN1 and Hsp70 have been confirmed to participate in PCV2-induced apoptosis.

Apoptosis Regulated by Cap and MKRN1
According to Gray et al. (2000) and Lee et al. (2013), MKRN1 is a transcriptional co-regulator and an E3 ubiquitin ligase that is highly evolutionarily conserved in vertebrates, it can also mediate apoptosis and p53-dependent cell cycle arrest. The interactions between different types of PCVs and their hosts have been analyzed by Finsterbusch's group, the research demonstrated that MKRN1 can interact with Cap proteins of both PCV1 and PCV2, resulting a decreased concentration of MKRN1 in the host (Finsterbusch et al., 2009a). The decreased MKRN1 can in turn reduce the level of p53 ubiquitination, resulting in an increase of p53 and thus promote cellular apoptosis. Under normal conditions, p53 and p21 both are suppressed by MKRN1 through ubiquitin-dependent degradation (Lee et al., 2009).
Previous studies have showed that p21 is capable of activating cell cycle arrest via suppressing apoptosis (Gartel and Tyner, 2002;Javelaud and Besancon, 2002;Abbas and Dutta, 2009;Jung et al., 2010). Therefore, ubiquitination and degradation of p21 mediated by MKRN1 may also contribute to trigger apoptosis. However, under stresses such as DNA damage, only p53 is suppressed by MKRN1; p21, which is not ubiquitinated by MKRN1 can also inhibit p53. Therefore, when the suppression of p53 by MKRN1 and p21 is reduced, the concentration of p53 will increase and thus promotes the apoptotic process (Lee et al., 2009).

Apoptosis Regulated by Cap and Hsp70
Hsp70 is a chaperone whose expression is induced by a variety of stimuli. A previous study confirmed that Hsp70 could inhibit the production of apoptosome and apoptosis in varying degrees by suppressing the activity of AIF (Garrido et al., 2006). In study of PMWS pathogenesis using proteomics strategies, Ramírez-Boo et al. (2011) reported that the down-regulation of Hsp70 was detected in inguinal lymph nodes of piglets after inoculation with PCV2. Another study regarding the interaction between PCV2 and target immune cells showed the expression of Hsp70 was up-regulated in PAMs during the initial stage of PCV2 infection (Liu et al., 2013a). A recent study showed that the PCV2 Cap protein can interact with Hsp70 and activate it further, then apoptosis could be inhibited via blockage the activation of caspase-3 in 3D4/31 cells (Liu et al., 2013b). However, the interaction between the PCV2 Cap protein and Hsp70 may reduce Hsp70 levels, and activate caspase-3 which plays a key role in the execution of apoptosis. Accordingly, Hsp70 might play different roles on different stages of PCV2 infection (Figure 1). For example, Hsp70 can be induced by PCV2 infection, but lack of sufficient Cap protein in the early stage of viral infection could result in inhibiting apoptosis by Hsp70. In contrast, during the later stages of PCV2 infection, the anti-apoptotic responses may be weakened by the down-regulation of Hsp70 levels due to the increased interaction with the PCV2 Cap protein.

ORF3 and Its Mechanisms of Apoptosis Regulation
Although the PCV2 non-structural protein ORF3 is not critical for viral replication Lin et al. (2013), found that the nuclear localization of ORF3 is correlated with triggering apoptotic response in porcine PBMC, it was also involved in PCV2induced the extrinsic apoptosis pathway through activation of the caspase8 and caspase3 (Kiupel et al., 2005;Liu et al., 2005). During a study of modulation of cellular functions by the PCV2 ORF3 protein, the ORF3 protein was found to directly interacted with pPirh2 (also called RCHY1), Pirh2 is an E3 ubiquitin ligase targeting p53 and leading p53 to degradation. The interaction between the pPirh2 and ORF3 protein could suppress pPirh2 stabilization and increase p53 cellular levels, thereby leading to apoptosis (Leng et al., 2003;Liu et al., 2007). Furthermore, present research suggests the amino acid residues of ORF3 protein are indispensable to compete with the interaction with pPirh2 over p53 (Timmusk et al., 2008;Karuppannan et al., 2010). p53 is a tumor suppressor as well as a transcription factor (Levine and Oren, 2009); it is also involved in regulation of diverse biological responses such as apoptosis, DNA damage, cell cycle arrest, oncogenic activities, erosion of telomeres, hypoxia and other physiological processes (Vousden and Prives, 2009;Collavin et al., 2010;Chang et al., 2013). It was reported that p53 was involved in apoptosis through transcription-dependent or -independent mechanisms during stress Xu et al., 2016). In general, the p53 protein content in cells is maintained at a very low level in the absence of stress through binding to proteins such as MDM2 (denoted HDM2 in humans), COP1, pPirh2 and JNK, which facilitates the degradation of p53 by the ubiquitin/proteasome pathway ( Table 2). In stress situations such as cell cycle arrest, apoptosis may be caused by a complex formed by p53 with pro-apoptotic and anti-apoptotic members of the Bcl-2 family (such as Bcl-2, Bcl-xL, Bak and Bax). Then, the complex triggers MOMP, liberating essential apoptotic factors (such as Cyt c, AIF, and Apaf-1) and ultimately causing a caspase cascade and apoptosis via the intrinsic pathway (Marchenko et al., 2007;Wolff et al., 2008;Green and Kroemer, 2009;Collavin et al., 2010).
Based on these works, induction of apoptosis during PCV2 infection is a complex process that may involve cross-talk between the intrinsic and the extrinsic apoptotic pathway. Certainly, the mechanistic role of PCV2 ORF3 protein in the regulation of apoptosis should be investigated in more detail in future studies.
To date, more than twenty proteins have been shown to be associated with pPirh2 (Jung et al., 2012). Additionally, p53 is a highly connected protein that could form physical complexes with a variety of cellular proteins (Collavin et al., 2010); currently, more than 320 reported interactions with human p53 are included in the APID web interface 1 , including kinases, phosphatases, acetyltransferases, de-acetylases, ubiquitin ligases, and other proteins. Accordingly, future investigation should consider whether there are other factors regulate PCV2induced apoptosis by participating in the interactions of pPirh2 and ORF3.

ORF4 and Its Mechanisms of Apoptosis Regulation
Studies indicated that the ORF4 protein is not required for PCV2 replication in mice or in PK-15 cells, while present research showed it plays a vital role in inhibiting apoptosis after PCV2 infection (He et al., 2013). Subsequently, Gao et al. (2014a) constructed two mutants of PCV2 ORF4: M1-PCV2 and M2-PCV2. By comparing the ORF3 mRNA levels of the wild-type and ORF4-deficient viruses in PK-15 cell, it was found that the ORF3 transcription levels of both ORF4 mutants were enhanced, indicating that the ORF4 protein may play an important role in preventing PCV2-induced apoptosis via inhibiting ORF3 transcription. Significant increases in caspase-8 and caspase-3 activities in both ORF4 mutants compared to wild-type PCV2 further confirmed this (Gao et al., 2014a). Subsequently Lv et al. (2016), revealed a mechanism by which ORF4 exerts cytoprotective function by resisting apoptosis in the early stage of PCV2 infection. Lv et al. (2015b) demonstrated the physical interaction between PCV2 ORF4 protein and FHC for the first time, and found that the decreased concentration of FHC can effectively inhibit the accumulation of reactive oxygen species FIGURE 2 | A hypothetical model describes the mechanisms involved in PCV2-ralated proteins induced apoptosis. On the one hand, Cap protein expressed by PCV2, which subsequently inhibits p53 and its downstream pro-apoptotic factors CytC, capases9, and caspase3 via MARNI pathway; it can also suppress Hsp70 and further inhibition the production of AIF and Apoptosome, depressing apoptosis. On the other hand, ORF3 and ORF4 proteins are largely involved in regulating apoptosis induced by PCV2: ORF3 protein interacts with pPirh2 to up-regulate the expression of p53 and its downstream factors to initiate apoptosis; whereas ORF4 protein inhibits apoptosis by suppressing activation of ORF3, it can also interact with FHC to reduce the content of FHC, inhibiting the production of ROS and ultimately suppression apoptosis.
in PCV2-infected cells, thereby inhibiting apoptosis. Recently, Lin et al. (2018) found that ORF4 is a mitochondrial targeting protein that ultimately induces apoptosis via the mitochondrial pathway by interacting with adenine nucleotide translocase 3 (ANT3). In summary, it is very significant to study how the apoptotic processes are regulated by the proteins of PCV2 to promote its infection (Figure 2). In addition to the factors mentioned above, there are other reported mechanisms that could regulate PCV2induced apoptosis, including different pathways (PERK/eIF2α, PI3K/Akt, and Fas/FasL), regulation of free Ca 2+ concentration and NF-κB activation. In the following sections, we will briefly review these factors.

PCV2-INDUCED APOPTOSIS REGULATED BY DIFFERENT PATHWAYS PERK/eIF2α Pathway
Mounting evidence indicates that a wide variety of viruses could disturb ER homeostasis and lead to ER stress (Li et al., 2015). To cope with this stress, cells evolve a series of adaptive mechanisms called the UPR (Hetz, 2012). ER stress activates three branches of the UPR: PERK (Shen et al., 2005), IRE1 (Chen and Brandizzi, 2013), and ATF6 (Yan et al., 2002). Zhou et al. (2016) demonstrated that PCV2 initiated UPR by activating the PERK/eIF2α pathway instead of IRE1 or ATF6 pathways, ultimately promoting viral replication in PK-15 cells (Figure 3). Since PERK/eIF2α further activates downstream factors ATF4 and CHOP, so PCV2 infection can selectively activate apoptosis via the PERK-eIF2α-ATF4-CHOP axis. The findings provide a basis for demonstrating that ER stress of apoptotic responses plays an important role in the pathogenesis of PCV2 infection.

PI3K/Akt and ASK1 Pathway
The phosphatidylinositol 3-kinase PI3K/Akt pathway plays a vital role in multiple physiological processes, such as inflammation suppression, carbohydrate metabolism, and cellular proliferation (Hsu et al., 2010). The PI3K/Akt pathway is also an indispensable target for a variety of viruses to inhibit apoptosis (Cooray, 2004;Shin et al., 2007;Soares et al., 2009). For example, PRRSV can trigger the PI3K/Akt pathway to augment viral replication and promote cell survival . Recently, Wei et al. (2012a) found that PCV2 can transiently activate the PI3K/Akt pathway, and the activated PI3K/Akt pathway could suppress premature apoptosis, thereby improving virus growth (Figure 3). However, in the early stage of PCV2 infection, inhibition of PI3K activation greatly enhanced apoptotic responses, mainly manifested by the cleavage of caspase3 and poly-ADP ribose polymerase as well as DNA fragmentation. The ASK1 plays a target role in the induction of apoptosis as an upstream enzyme that activates the p38/MARK and JNK pathways (Gan et al., 2016). During PCV2 infection, PI3K was activated first, followed by phosphorylation of Akt. Activated Akt inhibits the production of pro-apoptotic proteins such as JNK and p38/MAPK, thereby suppressing JNK-and p38-dependent apoptosis .
Interestingly, a previous study demonstrated that PCV2 infection regulates apoptosis by activating the p38/MAPK and JNK1/2 cellular stress pathways (Tibbles and Woodgett, 1999;Kyriakis and Avruch, 2001;Wada and Penninger, 2004;Wei et al., 2009). In the absence of stress, non-phosphorylated JNK bonds to p53, resulting in ubiquitination of p53 followed by proteasomal degradation (Fuchs et al., 1998a,b). In contrast, dissociation of p53 can be mediated by phosphorylated JNK, thus promoting p53 stabilization (Fuchs et al., 1998b). Additionally, p38/MAPK kinase plays a role not only in phosphorylation of p53 but also in transcription of p53-regulated Bax (Bulavin et al., 1999;Huang et al., 1999). Taken together, the activation and phosphorylation of p38/MARK and JNK after PCV2 infection might contribute to p53 stabilization, finally leading to apoptosis (Wei et al., 2009). Chang et al. (2007a) evaluated and compared the effects of infection of both PCV2 and PRRSV, individually or together, on co-cultured splenic (SLs), peripheral blood (PBLs) lymphocytes and swine splenic macrophages (SMs) in vitro. The expression FIGURE 3 | Summarizes multiply host cellular signaling pathways involved in regulating PCV2-induced apoptosis. First, PCV2 infection can activate PERK via PERK-eIF2α-ATF4-CHOP axis and then induce apoptosis, it can further activate JNK/p38 by activating the ASK1 pathway to ultimately promote apoptosis, whereas P13K/AKT plays an opposite role. Second, it can activate Cyt C and caspase-3 via the IP3R-1-Ca 2+ -PITC and NFκB-p53 pathways to activate apoptosis. In addition, PCV2 infection may activate caspase-8 via the Fas/FasL axis of the death receptor pathway to promote apoptosis. levels of Fas ligant (FasL) and Fas were significantly increased after PRRSV alone-and PCV2 and PRRSV dually inoculated groups, and the latter was more obvious, while increased Fas/FasL futher mediated apoptosis. Fas is also termed as CD95 (APO-1) and is one of the death receptors, these receptors include TNF-R1, CD95 (APO-1/Fas), DR3 (APO-3/TRAMP/Wsl-1/LARD), DR4 (TRAIL-R1), and so on. Han et al. (2010) confirmed that Fas could trigger apoptosis by binding to its cognate ligand, FasL. Thus, PCV2 infection may be associated with Fas/FasLmediated apoptosis (Figure 3). However, the hypothesis of the mechanism is still poorly understood and need to be further demonstrated.

NF-κB Pathway
The transcription factor NF-κB is commonly activated during viral infection and is a key molecule that regulates a variety of cellular signal transduction pathways (Bonizzi and Karin, 2004;Hayden and Ghosh, 2004). For example, Dengue virus, Reovirus, infectious bursal disease virus, Hepatitis B virus and Sindbis virus have been confirmed to trigger apoptosis via activating NF-κB Connolly et al., 2000;Jan et al., 2000;Liu and Vakharia, 2006;Pan et al., 2011;. In these processes, NF-κB serves as a pro-apoptotic factor which is able to activate the p53 signaling pathway (Fujioka et al., 2004).
The present study found that after PCV2 infected cells, NF-κB was activated simultaneously with viral replication, which was characterized by translocation of NF-κB from the cytoplasm to the nucleus, degradation and phosphorylation of IκBα protein and increased DNA binding activity. However, treatment of cells with CAPE, a selective inhibitor of NF-κB activation, reduced progeny production and virus protein expression followed by decreasing caspase activity, indicating the importance of NF-κB in inducing apoptosis (Wei et al., 2008). However, the exact details still to be further demonstrated. According to the above discussions, there are many factors and multiply pathways participate in regulating apoptosis induced by PCV2 (Figure 3).

PCV2-INDUCED APOPTOSIS REGULATED BY CALCIUM
Calcium ions (Ca 2+ ) are participated in multiple cellular physiological processes, such as cytoplasmic Ca 2+ signaling, ATP production, hormone metabolism and apoptosis induction (Drago et al., 2011). The intracellular free Ca 2+ ([Ca 2+ ]i) can activate apoptosis by regulating numerous calcium-sensitive enzymes and can also activate the mitochondrial apoptotic pathway via its accumulation in the mitochondria (Hajnoczky et al., 2003;Pathak et al., 2013). Lv et al. (2012) found that PCV2 could lead to apoptosis of lymphocytes, this apoptotic mechanism is affected by the increased [Ca 2+ ]i and is associated with the calmodulin (Lee et al., 2009) protein. Possible mechanisms of [Ca 2+ ]i induction include the suppression of Ca 2+ efflux by regulation of the Ca 2+ -ATPase transporter on cytomembranes, and/or the induction of Ca 2+ influx by promoting Ca 2+ release from the ER by increased expression of IP3R (Lv et al., 2012). IP3R can regulate the mobilization of Ca 2+ (Berridge, 2005), Ca 2+ released from the ER could activate the PTPC on mitochondria, causing Cyt c release and inducing apoptosis (Figure 3; Garrido et al., 2006).

CONCLUSION
Apoptosis is a very important host defense mechanism that contributes to remove infected, damaged and excess amounts of cells. The virus must evade host defense mechanisms to proliferate and spread. Infection with PCV2 has been demonstrated to trigger several signaling pathways such as PERK/eIF2α and PI3K/Akt pathway (Wei et al., 2012a;Zhou et al., 2016), resulting in activation or suppression of apoptosis. On the other hand, to cope with the apoptotic responses caused by viral infections, many viral proteins interact with apoptotic signals molecules to regulate apoptosis. There may be a discrepancy between induction and inhibition of apoptosis after PCV2 infection, as the experimental situation can be different, and close relationships between apoptosis and other factors that regulate cell fate, such as Ca 2+ , can make it more complicated and difficult.
This review is the first glimpse of PCV2 infection-induced apoptosis based on a wide array of reported works concerning PCV2 infection. It summarized currently findings which are involved in PCV2 infection-induced apoptosis, containing a vast panel of distinct pro-apoptotic and anti-apoptotic mechanisms (Figure 4). In the future, more attention should be taken on host-virus interaction. Further investigation that effect of different isoforms of PCV2, PCV1, and PCV3 on PCVinduced apoptosis should be done. Taking the above ideas into consideration will help us reach a deeper understanding of the molecular mechanisms of PCV2-induced apoptosis and open a new gate for further studies on the pathogenesis of PCV2.

AUTHOR CONTRIBUTIONS
PL and RJ contributed ideas for the review. YP wrote the manuscript and produced the figures. RJ, MW, ZY, and AC edited and revised the manuscript.