Coronavirus PLP2 and p53: inactivation by MDM2 and implications for apoptosis

As discussed before, various viral proteins localise to the ER and modulate the ER stress response, including inducing the expression of ER resident chaperones and proteins involved in autophagy thus promoting cell survival. Briefly, the accumulation of unfolded proteins in the ER lumen sequentially activates three pathways by activating three sensors -PERK, ATF6, and IRE1- each of which induce the expression of chaperones and other enzymes involved in the folding of proteins as well as activating autophagy by dephosphorylating Bcl-2 and inducing the expression of autophagy related genes, thus not only allowing folding of proteins but also degradation of misfolded proteins via autophagy. Persistent ER stress however induces apoptosis probably via activation of ER resident caspase-12 and activation of the intrinsic apoptotic pathway via DRAM-5 in a CHOP dependent manner. In this model, ATF6 is a transcription factor inducing the expression of genes associated with chaperones, whereas both PERK and IRE1 are protein kinases involved in inhibiting translation by phosphorylating eIF2α and processing the mRNA of XBP1, leading to the accumulation and induction of sXBP1 which in turn is a transcription specific for genes encoding chaperones.

Interestingly the induction of ER stress in human diploid WI-38, A549 or human fibrosarcoma HT1080 cells by MG132, thapsigargin and tunicamycin treatment as well as glucose deprivation induces the accumulation of p53 in the cytoplasm as well as destabilization of p53, indicating the phosphorylation of p53 at Ser-315/Ser-376 by Glycogen Synthase Kinase 3β (GSK-3β) as well as binding of Hdm2 (the human equivalent of MDM2). Indeed, under ER stress conditions a complex of p53 with both Hdm2 and GSK-3β forms, although during prolonged ER stress levels of functional Hdm2 decrease, thus inducing p53 dependent apoptosis.

In the opinion of the author of this post, it seems therefore possible that the expression of coronaviral proteins at least initially protects infected cells from undergoing apoptosis by the induction of a cytoprotective ER stress response involving the phosphorylation of p53 in a GSK-3β dependent manner. It should be noted however that the phosphorylation and activation of GSK-3β in Vero E6 cells infected with SARS-CoV fails to protect cells from apoptosis at 18 h p.i. ; to my knowledge however earlier timepoints have not been tested.

In the case of Porcine Respiratory Syndrome Virus (PRRSV), a member of the Arteriviridae, the phosphorylation of p53 via Akt kinase by Nutlin-3 protects PRRSV infected Marc-145 from apoptosis and promotes viral replication as measured at 48 hrs p.i., whereas p53 inactivation by PFT decreases viral replication (at 24 hrs p.i.). Although the precise mechanism has not been identified, it has been proposed that PRRSV mediated activation of p53 leads to  inhibition of c-Jun N-terminal kinase (JNK). Since JNK is also induced as a result of activating IRE1 and in the phosphorylation of ER resident Bcl-2 it would be interesting to not only investigate if PRRSV infection stabilises the Beclin-1/Bcl-2 complex at the ER but also compare PRRSV to Coronaviruses - in addition to identify the viral protein(s) involved, with the nsp567 polyprotein of PRRSV being a strong candidate. Interestingly, PRRSV nsp567 induces the formation of autophagy-like vesicles akin to CoV nsp-6 in HEK-293T cells.

PRRSV and UPR: activation of IRE1 signalling early in infection, inhibition
late in infection?

Coronavirus PLP and p53: inhibition of antiviral signalling

Coronaviruses are associated (mainly) with relatively benign infections in humans of the respiratory, hepatic, enteric, and central nervous system, with the recently emerged Severe and Acute Respiratory Syndrome (SARS)-CoV and Middle Eastern Respiratory Syndrome (MERS)-CoV as well the Human Coronavirus NL-63 (HCoV-NL63) being the exception. A central role in the formation of the viral replication centers is the formation of double membrane vesicles that utilizes the cellular autophagy machinery. The degradation of autophagosomes by fusion with the lysosome however is inhibited by viral non-structural proteins (nsp), including nsp-6 as well as the viral proteases, PLP2 and PL^Pro respectively, via inhibiting enzymes required for the maturation of the lysosome (in the case of nsp-6) or fusion with the lysosome (in the case of PLP2/PL^Pro).  Both PLP2/PL^Pro     derived from MHV-A59, SARS-CoV, and HCoV-NL63 have also been shown to inhibit antiviral signalling by antagonizing STING induced activation of IFN following treatment of cells with Poly(I:C) in the absence of other viral proteins, suggesting that expression of the viral protease is sufficient to  inactivate STING mediated signalling.  Since STING mediated signalling involves K-63 ubiquitination of STING prior to its association with TBK-1 and IRF-3, it has been proposed that PLP2/PL^Pro deubiquitinates STING as well as TBK-1, RIG-1, and IRF-3 via the Deubiquitinase domain (DUB) as well as deISGylating cellular proteins involved in antiviral signalling. 

The expression of IFN-β in particular can also be activated in a p53 dependent manner by inducing the expression of two IFN regulatory factors, IRF-7 and -9.  Transfection of renal carcinoma cells (RCC) with Poly(I:C) accordingly not only increased the levels of phosphorylated p53, NOXA, and tBid -and thus inducing apoptosis- but also increases the mRNA levels of IFN-β in a TLR-3  as well as 2-5OAS and RNaseL  dependent manner.

2,5-OAS mediated signalling pathway

The stability of p53 is negatively regulated by MDM2, a p53-specific E3 Ubiquitin ligase that ubiquitinylates p53 and thus induces the proteasome mediated degradation of p53 in the cytoplasm as well as inhibiting the transcriptional activity of p53. In non-infected cells, MDM2 is located in the cytoplasm and ubiquitinylated, thus being inactive (due to degradation) and stabilised by a cellular homologue of HAUSP. Deubiqutinated MDM2 however translocates to the nucleus where it binds to Ser-315/Ser-376 phosphorylated p53. This complex then translocates into the cytoplasm where ubiqutinated p53 is degraded. In order to deactivate p53 and thus p53-dependent antiviral signalling, at least two conditions have to be met: (1) MDM2 (or Hdm2) has to be deubiqutinated and (2) p53 has to be phosphorylated at Ser-315/Ser-376.  In cells infected with Coronavirus’ both conditions are met since the induction of the ER stress response induces the phosphorylation of p53 in a GSK-3β dependent manner as described above and the Coronavirus genome encodes with the viral PLP2/PL^Pro a protein that has a DUB.

Indeed, Porcine epidemic diarrhoea virus (PEDV) derived PLP2 has been shown to stabilise and to deubiquitinate exogenous Hdm2 in p53^+/+ HCT cells whilst increasing the degradation of p53 via the ubiquitin-dependent proteasome pathway as well as inhibiting IFN-β induced expression of a luciferase reporter gene following transfection of Poly(I:C) concomitant with nuclear translocation of Hdm2. Accordingly, p53 activity following transfection with Poly(I:C)  is increased in p53 ^-/- HCT cells irrespective of PLP2 . Furthermore, in p53 ^-/- HCT cells PLP2 fails to protect cells from apoptotic cell death induced by PUMA, indicating that the expression of PLP2 induces PUMA expression via p53. Indeed PUMA has been shown to cause apoptosis as a result of ER stress suggesting that the expression of PLP2 induces ER stress; if this is due to the increase in the formation of autophagosomes remains to be seen.

PLP2 mediated translocation of MDM2 via binding to DUB

In conclusion, the expression of PEDV PLP2 induces not only the ER stress but also inactivates p53 mediated activation of antiviral signalling following the transfection of p53 WT HCT cells with Poly(I:C) (and thus presumably also by viral RNA activated signalling) by deubiquitinating the human homologue of MDM2, Hdm2, and subsequent degradation of p53, thus blocking the type I Interferon response as well as preventing PUMA dependent apoptosis. In this context it is interesting that the infection of cells with Influenza Virus A induces the activation of the type I Interferon via p53; in contrast to the coronaviral PLP2 however, Influenza A Virus does not antagonize p53 mediated antiviral signalling.  Since the coronaviral PLP2 increases the replication of Sendai Virus in p53^+/+ MEF, it seems conceivable that in cells expressing PLP2 Influenza A Virus replication is also increased.




From a therapeutically point of view it might be interesting to investigate if mice deficient for MDM2 or treated with small molecule inhibitors of MDM2 are more susceptible to Coronavirus mediated infections.

Finally, the degradation of p53 by PLP2 might also prevent the induction of the phagophore. p53 not only transactivates the expression of pro-inflammatory and pro-apoptotic genes but also of genes facilitating the induction of the phagophore, including DRAM-1.

p53 and autophagy induction

Expression of PLP2 therefore might inhibit this pathway as well; since some of those genes whose expression is induced are not only inducing the formation of the phagophore but also connecting autophagy induction to apoptosis, repressing p53 mediated signalling might also affect autophagy induced apoptosis, particularly in a situation where the fusion of autophagosomes with the lysosome in inhibited (as it is in cells expressing nsp-6 or PLP2).  

PLP2 and p53 mediated activation of autophagy: consequences for autophagy
related apoptosis?

PLP2 and autophagy: multiple points of interference

Further reading

Pluquet O, Qu LK, Baltzis D, & Koromilas AE (2005). Endoplasmic reticulum stress accelerates p53 degradation by the cooperative actions of Hdm2 and glycogen synthase kinase 3beta. Molecular and cellular biology, 25 (21), 9392-405 PMID: 16227590 

  
Li K, Chen Z, Kato N, Gale M Jr, & Lemon SM (2005). Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. The Journal of biological chemistry, 280 (17), 16739-47 PMID: 15737993 

Harashima N, Minami T, Uemura H, & Harada M (2014). Transfection of poly(I:C) can induce reactive oxygen species-triggered apoptosis and interferon-β-mediated growth arrest in human renal cell carcinoma cells via innate adjuvant receptors and the 2-5A system. Molecular cancer, 13 PMID: 25227113 

Matsumoto M, & Seya T (2008). TLR3: interferon induction by double-stranded RNA including poly(I:C). Advanced drug delivery reviews, 60 (7), 805-12 PMID: 18262679 

Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J, & Gu W (2002). Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature, 416 (6881), 648-53 PMID: 11923872

Li M, Brooks CL, Kon N, & Gu W (2004). A dynamic role of HAUSP in the p53-Mdm2 pathway. Molecular cell, 13 (6), 879-86 PMID: 15053880 

Kon N, Kobayashi Y, Li M, Brooks CL, Ludwig T, & Gu W (2010). Inactivation of HAUSP in vivo modulates p53 function. Oncogene, 29 (9), 1270-9 PMID: 19946331

Brooks CL, & Gu W (2004). Dynamics in the p53-Mdm2 ubiquitination pathway. Cell cycle (Georgetown, Tex.), 3 (7), 895-9 PMID: 15254415 
  
Lu M, Xia L, Li Y, Wang X, & Hoffman R (2014). The orally bioavailable MDM2 antagonist RG7112 and pegylated interferon α 2a target JAK2V617F-positive progenitor and stem cells. Blood, 124 (5), 771-9 PMID: 24869939 
  
Mizutani T, Fukushi S, Saijo M, Kurane I, & Morikawa S (2004). Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells. Virology, 327 (2), 169-74 PMID: 15351204 

Wang X, Zhang H, Abel AM, Young AJ, Xie L, & Xie Z (2014). Role of phosphatidylinositol 3-kinase (PI3K) and Akt1 kinase in porcine reproductive and respiratory syndrome virus (PRRSV) replication. Archives of virology, 159 (8), 2091-6 PMID: 24532302 
  
Fung TS, Huang M, & Liu DX (2014). Coronavirus-induced ER stress response and its involvement in regulation of coronavirus-host interactions. Virus research, 194, 110-23 PMID: 25304691 

Cottam EM, Maier HJ, Manifava M, Vaux LC, Chandra-Schoenfelder P, Gerner W, Britton P, Ktistakis NT, & Wileman T (2011). Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy, 7 (11), 1335-47 PMID: 21799305 
  
Huo Y, Fan L, Yin S, Dong Y, Guo X, Yang H, & Hu H (2013). Involvement of unfolded protein response, p53 and Akt in modulation of porcine reproductive and respiratory syndrome virus-mediated JNK activation. Virology, 444 (1-2), 233-40 PMID: 23850458 

DeDiego ML, Nieto-Torres JL, Regla-Nava JA, Jimenez-Guardeño JM, Fernandez-Delgado R, Fett C, Castaño-Rodriguez C, Perlman S, & Enjuanes L (2014). Inhibition of NF-κB-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival. Journal of virology, 88 (2), 913-24 PMID: 24198408 
  
Yuan L, Chen Z, Song S, Wang S, Tian C, Xing G, Chen X, Xiao ZX, He F, & Zhang L (2014). p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling. The Journal of biological chemistry PMID: 25505178 
  
Tan S, Wei X, Song M, Tao J, Yang Y, Khatoon S, Liu H, Jiang J, & Wu B (2014). PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy. Cell death & disease, 5 PMID: 24625987 
  
Zhao Y, Yu S, Sun W, Liu L, Lu J, McEachern D, Shargary S, Bernard D, Li X, Zhao T, Zou P, Sun D, & Wang S (2013). A potent small-molecule inhibitor of the MDM2-p53 interaction (MI-888) achieved complete and durable tumor regression in mice. Journal of medicinal chemistry, 56 (13), 5553-61 PMID: 23786219

Shin SW, Kim SY, & Park JW (2012). Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells. Biochimica et biophysica acta, 1823 (2), 451-7 PMID: 22178132

Peng M, Yin N, & Li MO (2014). Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling. Cell, 159 (1), 122-33 PMID: 25259925

Coxsackievirus B3 and BPIFB3: silencing required for viral replication?

Coxsackievirus B3 (CVB3) is a positive strand RNA virus with a non-segmented genome of approx. 7.4 kB in size, encoding a single polyprotein which is cleaved by cellular and viral proteins to generate the non-structural and structural proteins. As discussed in a previous post, following the infection of pancreatic acinar cells with CVB3 autophagosome-like vesicles can be observed in infected cells. Akin to the role of the induction of autophagy in the formation of replication centers following the infection of cells with Corona- or Arterivirus’, the autophagy machinery is required for forming the replication centers whilst at the same time the degradation of autophagosomes containing components of the viral replication complex via fusion with the lysosome and subsequent formation of the autolysosome is inhibited. Indeed, in pancreatic acinar cells infected with CVB3, “megaphagosomes” containing components of the viral replication complex can be observed in the absence of increased autophagic flux. Since the application of 3-Methyladenine (3-MA) or siRNAs targeting components of the autophagic machinery such as Beclin-1, Vps34, Atg5, or Atg7 not only inhibits the formation of megaphagosomes but also viral replication and the induction of autophagy by Rapamycin or starvation increases the number of megaphagosomes as well as viral titers, the induction of autophagosome formation has been proposed to be essential for CVB3 replication. Since these structures are located in close proximity to lysosomes it has been postulated that CVB3 inhibits the fusion of autophagosomes with the lysosome whilst inducing the formation of autophagosomes either by sequestering of proteins required for the initiation of phagophore formation or inducing autophagy either as a consequence of the localisation of viral proteins or by r (potentially) by cleavage of p62/SQSTM1 to the ER and thus by initiating the ER stress response as discussed before.

Model of Coxsackievirus B3 induced induction of autophagy via the induction of the
ER stress response

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) was recently identified by RNAi screening whose depletion enhances the replication of CVB3 but not Poliovirus in human brain microvascular endothelial cells (HBMEC) as determined by plaque assay of supernatants from infected cells. In contrast to cells treated with siBPIFB3, U2OS cells transfected with a plasmid allowing the overexpression of a C-terminal Flag tagged version of BPIFB3, however exhibited a decrease in viral titres, suggesting that BPIFB3 is required for efficient viral replication.

BPIFB3 domains and anchoring in the ER 

Unlike the rat homologue of BPIFB3, Rya3, human BPIFB3 localises to the ER in uninfected cells with a boomerang like structure in which the BIP1-fold 1 and -2 are interacting with the lipids and the C terminal exposed to the cytosol, although the precise structure is not known. Being a ER resident protein, BPIFB3 might either inhibit or promote autophagy by interacting with components of the autophagy machinery in particular Beclin-1.  Indeed, silencing BPIFB3 enhances both basal and starvation induced autophagy in HBMEC, HeLa as well as human kidney 786-O cells, suggesting that BPIFB3 binds proteins like Beclin-1 akin to Bcl-2 although this has not been shown yet. In contrast to BPIFB3 silencing, the overexpression of BPIFB3 induces the formation of large LC3B negative structures in the presence of overexpressed LC3 that are negative for both LAMP1 and BPIFB3, and thus are non-degradative. Given that these vacuole-like structures are negative for LC3B, their movement is restricted following the treatment of transfected cells with Rapamycin since they cannot associate with microtubuli. Interesting, these structures are also negative for p62/SQSTM1, suggesting that p62/SQSTM1 dependent selective autophagy might be impaired, although p62/SQSTM1 levels are not reduced in cells transfected with siBPIFB3. Concomitant to the formation of LC3B negative vacuoles, the conversion of LC3-I to LC3-II is inhibited probably by inhibition of the Atg4B protease and thus prevents the cleavage of LC3-I and subsequent conjugation.  From a structural point of view, the expression of BPI-1 fold was sufficient to induce the formation of these vacuoles and also co-localises with LC3B both in mock and Rapamycin treated cells.

Since the infection of pancreatic acinar cells with CVB3 has been associated with the formation of megaphagosomes, it might be possible that the expression of siBPFB3 increases the formation of megaphagosomes following CVB3 infection and increases viral replication. Indeed, BPFB3 silencing promotes the formation of megaphagosomes in HBMC infected with CVB3 but not in Poliovirus infected cells, as well as increasing the association of LC3B with viral replication complexes, similar to non-infected cells treated with siBPFB3.

Model of CVB3 induced autophagy in the presence and absence of BPIFB3: smaller
vesicles are induced in the presence of BPFB3 which may or may not contain viral RNA

It remains to be seen if the accumulation of viral proteins at the ER induces the degradation of BPIFIB3 and thus favours the formation of megaphagosomes or if viral proteins inhibit the inhibitory function of BPIFB3 by either binding BPIFB3 directly or by decreasing BPIFB3 expression. Alternatively, the viral proteins might target BPIFB3 for either proteasome dependent or independent degradation and thus favour the formation of megaphagosomes.

Further reading

Coyne CB, Bozym R, Morosky SA, Hanna SL, Mukherjee A, Tudor M, Kim KS, & Cherry S (2011). Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers. Cell host & microbe, 9 (1), 70-82 PMID: 21238948 

Delorme-Axford E, Morosky S, Bomberger J, Stolz DB, Jackson WT, & Coyne CB (2014). BPIFB3 regulates autophagy and coxsackievirus B replication through a noncanonical pathway independent of the core initiation machinery. mBio, 5 (6) PMID: 25491355 

Alirezaei M, Flynn CT, Wood MR, & Whitton JL (2012). Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo. Cell host & microbe, 11 (3), 298-305 PMID: 22423969 

Kemball CC, Alirezaei M, Flynn CT, Wood MR, Harkins S, Kiosses WB, & Whitton JL (2010). Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo. Journal of virology, 84 (23), 12110-24 PMID: 20861268