Virology tidbits

Virology tidbits

Monday, 29 December 2014

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 PLPro 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/PLPro).  Both PLP2/PLPro     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/PLPro 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/PLPro 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

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