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