Virus Research 185 (2014) 10–22

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Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain András Balogh a,b,1 , Judit Bátor a,b,1 , Lajos Markó c , Mária Németh a,b , Marianna Pap a,b , György Sétáló Jr. a,b , Dominik N. Müller c , Laszlo K. Csatary d , József Szeberényi a,b,∗ a

Department of Medical Biology, University of Pécs Medical School, Pécs, Hungary Signal Transduction Research Group, János Szentágothai Research Centre, Pécs H-7624, Hungary c Experimental and Clinical Research Center, Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine, Berlin 13125, Germany d United Cancer Research Institution, Alexandria 22307, VA, USA b

a r t i c l e

i n f o

Article history: Received 9 January 2014 Received in revised form 10 February 2014 Accepted 6 March 2014 Available online 15 March 2014 Keywords: Oncolytic virus Virotherapy Gene expression profiling Interferon signaling Apoptosis

a b s t r a c t Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12 h at a titer corresponding to the IC50 value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/Hinduced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The oncolytic potential of certain viruses has been known for a century. First they appeared as agents causing rare anecdotal miracles of hardly explainable recovery from malignant tumor (reviewed in Kelly and Russell, 2007), but later scientific evidence

∗ Corresponding author at: Department of Medical Biology, University of Pécs Medical School, H-7624 Pécs, Szigeti 12, Hungary. Tel.: +36 72 536 216; fax: +36 72 536 453. E-mail addresses: [email protected] (A. Balogh), [email protected] (J. Bátor), [email protected] (L. Markó), [email protected] (M. Németh), [email protected] (M. Pap), [email protected] (G. Sétáló Jr.), [email protected] (D.N. Müller), [email protected] (L.K. Csatary), [email protected] (J. Szeberényi). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.virusres.2014.03.003 0168-1702/© 2014 Elsevier B.V. All rights reserved.

has accumulated supporting the selective cytotoxicity of several viruses toward tumor cells (Eager and Nemunaitis, 2011). At present dozens of natural and genetically engineered viruses are being tested in pre-clinical and clinical trials presenting oncolytic virotherapy as a viable alternative to traditional cancer treatments. Newcastle disease virus (NDV) was among the first viruses whose oncolytic capability was suspected (Cassel and Garrett, 1965; Csatary, 1971; Wheelock and Dingle, 1964). NDV is an avian virus that, based on its pathogenicity in its natural hosts, can be classified into three pathotypes: velogenic (highly virulent), mesogenic (moderately pathogenic) and lentogenic (non-virulent) strains. NDV is an enveloped RNA virus belonging to the Paramyxoviridae family (for a review see Lech and Russell, 2010). It has several features that make it a promising natural agent for virotherapy: (I) it shows selective cytotoxicity toward tumor cells both in culture and in vivo (Fabian et al., 2007; Lorence et al., 2007); (II) even velogenic NDV strains have not been shown to cause human

A. Balogh et al. / Virus Research 185 (2014) 10–22

disease (except for mild flu-like conditions); (III) NDV is genetically stable not showing signs of antigenic drift or recombination; (IV) being an RNA virus lacking a DNA intermediate of replication, insertional mutagenesis as a side-effect can be ruled out. Despite extensive studies, the mechanism of oncolysis by NDV is still not fully understood: both immune stimulation and direct cytotoxicity may be involved (Schirrmacher et al., 1999). Individual case studies, phase I and II clinical trials were conducted using several NDV isolates (Eager and Nemunaitis, 2011; Lech and Russell, 2010). Systemic administration of the mesogenic NDV strains PV701 (Lorence et al., 2007) and MTH-68/H (Csatary et al., 2004) and the lentogenic isolate NDV-HUJ (Freeman et al., 2006) to patients with advanced cancer gave promising results. At present there is no explanation why some cancer patients respond well, while others are resistant to the oncolytic virotherapy by NDV vaccines. MTH-68/H, the NDV strain used in the present study, is an attenuated mesogenic virus. It displayed selective cytotoxicity toward rodent and human tumor cell lines killing them by apoptosis (Fabian et al., 2001, 2007), while non-transformed fibroblasts were found resistant to MTH-68/H infection. Analysis of the effects of MTH-68/H in the PC12 rat pheochromocytoma cell line revealed that (I) MTH-68/H replicated in PC12 cells and produced infectious progeny particles; (II) the apoptotic effect of MTH-68/H did not require the involvement of p53 protein; (III) MTH-68/H infection induced endoplasmic reticulum (ER) stress (Fabian et al., 2007). Although all tumor cell lines tested were sensitive to NDV infection they displayed a wide range of susceptibility to the virus (Fabian et al., 2007). In order to identify genes whose expression is altered in tumor cells during infection with MTH-68/H and therefore may be involved in oncolysis, genome-wide expression profiling was performed in this study. We selected the PC12 cell line, since these cells are killed by MTH-68/H, but they have relatively low sensitivity (IC50 value: 12.87 (Balogh et al., 2011); IC50 values for other tumor cell lines infected with MTH-68/H ranged from 0.01 to 70 [unpublished observations]) and the apoptotic events are relatively slow, giving time for kinetic analysis of cellular changes evoked by the virus. In addition, the moderate sensitivity of the PC12 cell line to MTH-68/H suggested to us that these cells, before undergoing apoptosis, may generate a relatively strong antiviral response to the virus thereby increasing the range of candidate NDV susceptibility genes. Based on previous analysis of key signaling proteins (Fabian et al., 2007) we choose a 12-h exposure to MTH-68/H for gene expression profiling. At this time point the cells are still alive, but irreversible changes in stress and apoptotic signaling have already taken place. We report here that upon MTH-68/H infection, hundreds of specific genes are up- or downregulated in PC12 cells. The possible significance of some of these gene expression changes in the oncolytic action of MTH-68/H is discussed. 2. Materials and methods 2.1. Cell culture and virus infection PC12 cells (Greene and Tischler, 1976; kindly provided by G.M. Cooper) were grown as described by Fabian et al. (2007). Cell cultures (106 cells in 60-mm plates) were infected with the NDV strain MTH-68/H (described by Fabian et al., 2007) at the IC50 value for 12 h. 2.2. Exon chip analysis Total cytoplasmic RNA from control and MTH-68/H-infected PC12 cell culture triplicates was isolated using Qiagen’s (Hilden,

11

Germany) RNeasy kit. Samples were analyzed using Affymetrix GeneChip Rat Exon 1.0 ST Array chip (Santa Clara, CA) by UDGenomed Ltd. (Debrecen, Hungary). Expression of specific genes was determined from raw microarray data. Gene expression data were normalized and the absolute fold-change expression was determined. At least 2-fold increase or decrease in expression was considered to be significant using unpaired T-probe with Benjamini-Hochberg correction. Functional categorization of genes with altered expression was performed by the DAVID functional annotation clustering tool (Huang et al., 2009). 2.3. RNA extraction and quantitative reverse transcriptase PCR Total RNA was isolated using the RNeasy kit (Qiagen). Concentration of isolated RNA was measured by NanoDrop-1000 spectrophotometer (Thermo Scientific, Waltham, MA). RNA samples possessed A260/A280 values greater than 1.8 and were considered to be pure for further analysis. Two micrograms of total RNA were used for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). SYBR Green or TaqMan analysis was conducted in duplicate using an Applied Biosystems 7700 Sequence Detector (Life Technologies), following the manufacturer’s protocols. The expression levels of the target genes were normalized to 18S rRNA levels and were calculated using the standard curve method. Primers and probes were designed using Primer Express software 3.0 (Applied Biosystems, Carlsbad, CA) and were synthesized by Biotez (Berlin, Germany). Primer and probe sequences are listed in Supplementary Table S3. Supplementary Table S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.03.003. 3. Results RNA samples isolated from control and MTH-68/H-infected PC12 cells were subjected to transcriptional profiling as described in Section 2. 729 genes (corresponding to 773 exons) were found to be induced and 612 genes (631 exons) to be repressed by virus infection, at least two-fold (see Supplementary Table S1 and S2). Transcriptional regulation of 5 of the up-regulated genes (Ifnb1, Stat2, Casp12, Ddx58 and Tnf) and 4 of the down-regulated genes (Bmyc, Mrpl34, Pole2, Cdc26) was validated by quantitative reverse transcriptase PCR (Supplementary Fig. S1). The tendency of regulation by MTH-68/H was found similar using the two different experimental approaches. Supplementary Fig. I related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2014.03.003. Analysis of the effect of MTH-68/H infection on elements of several regulatory networks of special interest is presented below. 3.1. Pathways leading to the secretion of type I interferons The antiviral strategy of cells depends on their innate immune response to viral infection leading to the secretion of type I interferons (IFNs), IFN␣ and ␤ (Sadler and Williams, 2008; Samuel, 2001). Viral components are recognized by cytosolic and transmembrane pattern recognition receptors (PRRs), their activation triggers distinct but interconnected signaling pathways ultimately stimulating the transcription of IFN-coding genes. 3.1.1. RIG-I-like receptor signaling The most important cytosolic antiviral PRR proteins belong to the family of RIG-I-like receptors (RLR; for abbreviations of all signaling proteins and their genes see Tables 1–3 ). RLRs are RNA helicases that bind various viral components and their activation

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A. Balogh et al. / Virus Research 185 (2014) 10–22

Table 1 The effect of MTH-68/H infection on the expression of genes involved in the induction of type I IFN genes in PC12 cells. Gene/protein Genes of RIG-I-like receptor pathways 10869879 Similar to IFN␣ 8/6 precursor Ddx60 DEAD box polypeptide 60 Ifnb1 Ifih1 (MDA5)

IFN ␤1 IFN-induced with helicase C domain 1

Irf7 Ddx58

IFN regulatory factor 7 (RIG-I) DEAD box polypeptide 58

Ifnb2 Dhx58

(IL6) IFN ␤2 (interleukin 6) (LGP2) DEXH box polypeptide 58

Trim25 Ifna11 Ifna4 Ifna1 Mavs (IPS-1)

Tripartite motif-containing 25 IFN ␣11 IFN ␣4 IFN ␣1 Mitochondrial antiviral signal adapter

Traf3

TNF receptor-associated factor 3

Tank Ikbke Irf3 Tbk1 Traf6

TRAF-associated NF␬B activator I␬B kinase ␧ IFN regulatory factor 3 TANK-binding kinase 1 TNF receptor-associated factor 6

Genes of Toll-like receptor pathways NF␬B inhibitor ␧ Nfkbie Nfkbiz NF␬B inhibitor z Tlr3 Toll-like receptor 3 Nfkbid NF␬B inhibitor ␦ Nfkbia NF␬B inhibitor ␣ Nuclear factor ␬B2 (NF␬B2) Nfkb2 Nfkb1 Nuclear factor ␬B1 (NF␬B1) MyD88 Myeloid differentiation early response gene 88 Nfkbib NF␬B inhibitor ␤ Ikbkb (IKK␤) I␬B kinase ␤ Ikbkg (NEMO) I␬B kinase ␥ (NF␬B essential modulator) Traf6 TNF receptor-associated factor 6 Irak1 Irak4 Ripk1 Traf3

IL-1 receptor-activated kinase 1 IL-1 receptor-activated kinase 4 Receptor-interacting serine-threonine kinase 1 TNF receptor-associated factor 3

Trif Tak1 Ikbka Tlr7/8 Tlr9 Tab1 Tab3

TIR-domain containing adapter inducing IFN␤ TGF␤-activated kinase 1 I␬B kinase ␣ Toll-like receptor 7/8 Toll-like receptor 9 TAK1 binding protein 1 TAK1 binding protein 3

a b c d

Protein function

Induction/repression by MTH-68/H (fold)a , b

References

Cytokine (putative) Cytosolic viral RNA receptor/helicase Cytokine/type I IFN Cytosolic viral RNA receptor/helicase (melanoma differentiation-associated gene 5) Transcription factor Cytosolic viral RNA receptor/helicase (retinoic acid inducible gene-I) Cytokine Cytosolic viral RNA receptor/helicase (laboratory of genetics and physiology 2) E3 ubiquitin/ISG15 ligase Cytokine/type I IFN Cytokine/type I IFN Cytokine/type I IFN Adapter protein (IFN␤ stimulator protein-1) E3 ubiquitin ligase/adapter protein Adapter protein Serine-threonine kinase Transcription factor Serine-threonine kinase E3 ubiquitin ligase/adapter protein

80.3↑ 67.3↑

Sadler and Williams (2008) Schoggins et al., 2011

49.5↑ 36.6↑

Sadler and Williams (2008) Richards and Macdonald (2011)

23.5↑ 18.8↑

Savitsky et al. (2010) Richards and Macdonald (2011)

15.2↑ 10.7↑

Sadler and Williams (2008) Richards and Macdonald (2011)

6.2↑ 4.0↑ 3.6↑ 3.4↑ NSAc

Richards and Macdonald (2011) Sadler and Williams (2008) Sadler and Williams (2008) Sadler and Williams (2008) Richards and Macdonald (2011)

NSA

Richards and Macdonald (2011)

NSA NSA NSA NSA NSA

Richards and Macdonald (2011) Richards and Macdonald (2011) Savitsky et al. (2010) Richards and Macdonald (2011) Richards and Macdonald (2011)

8.1↑ 6.0↑ 4.1↑ 3.6↑ 3.3↑ 3.0↑ 2.8↑ 2.1↑ 2.0↑ NSA NSA NSA

Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011)

NSA NSA NSA NSA

Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011)

NSA NSA NSA NDd ND ND ND

Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011) Richards and Macdonald (2011)

I␬B␧ I␬B z Transmembrane receptor I␬B␦ I␬B␣ Transcription factor Transcription factor Adapter protein I␬B ␤ Serine-threonine kinase Adapter protein E3 ubiquitin ligase/adapter protein Serine-threonine kinase Serine-threonine kinase Serine-threonine kinase E3 ubiquitin ligase/adapter protein Adapter protein Serine-threonine kinase Serine-threonine kinase Transmembrane receptor Transmembrane receptor Adapter protein Adapter protein

PC12 cells were infected with MTH-68/H at IC50 for 12 h. Steady-state levels of mRNAs were determined using oligonucleotide microarrays as described in Section 2. Induction and repression of genes are indicated by upward and downward arrows, respectively. Gene expression was not significantly altered (less than 2-fold induction or repression) by MTH-68/H. Gene expression was not detected by the microarray.

leads to RLR signaling (Richards and Macdonald, 2011; Fig. 1A). The genes of all four RLR proteins expressed in PC12 cells (RIG-I, MDA5, LGP2 and DDX60) were strongly induced by MTH-68/H infection (Table 1) presumably by IFN␣/␤ further amplifying the signal on the RLR pathway. RIG-I is polyubiquitinated and activated by TRIM25, an IFN-induced E3 ubiquitin ligase (Napolitano and Meroni, 2012). Trim25 was also induced in MTH-68/H-infected cells (Table 1). The

adapter proteins IPS-1, TRAF3, TRAF6 and TANK recruit IKK-related protein kinases TBK1 and IKK␧ to the RIG-I complex and, upon activation of these kinases, the transcription factors IRF3 and IRF7 are phosphorylated and translocated to the nucleus, whereby they induce the transcription of IFN␣ and IFN␤ coding genes (Savitsky et al., 2010). While Irf3 was constitutively expressed in PC12 cells, Irf7 was strongly induced by MTH-68/H infection (Table 1).

A. Balogh et al. / Virus Research 185 (2014) 10–22

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Table 2 Expression of genes involved in IFN-signaling in MTH-68/H-infected PC12 cells. Gene/protein Signaling to ISRE and GAS enhancer elements 10869879 Similar to IFN␣ 8/6 precursor Ifnb1 IFN ␤1 10720237 Similar to IFN ␭2

Irf9 Ifna11 Ifna4 Jak2

IFN ␤2 (interleukin 6) Signal transducer and activator of transcription 2 Signal transducer and activator of transcription 1 Interferon regulatory factor 9 IFN ␣11 IFN ␣4 Janus kinase 2

Ifna1 Ifngr1 Ifngr2 Ifnlr1 Il10r2

IFN ␣1 IFN ␥ receptor 1 IFN ␥ receptor 2 IFN ␭ receptor 1 IL10 receptor 2

Ifnar1 Jak1

IFN ␣ receptor 1 Janus kinase 1

Tyk2

Tyrosine kinase 2

Ifng Ifnar2

IFN ␥ IFN ␣ receptor 2

Ifnb2 (IL6) Stat2 Stat1

Type I IFN effector pathways ISGylation pathways IFN-stimulated gene 15 Isg15 Herc6 HECT domain and RLD containing protein 6 Usp18 Ubiquitin-specific peptidase 18 Uba7 Ubiquitin-activating enzyme 7 Trim25 Usp49 Ube2E1

Tripartite motif-containing protein 25 Ubiquitin-specific peptidase 49 Ubiquitin-conjugating enzyme E1

Ube2t

Ubiquitin-conjugating enzyme E2 T

Ube2L6

Ubiquitin-conjugating enzyme E2L6

Protein kinase R pathway Eukaryotic translation initiation Eif2a factor 2␣ Eif2ak (PKR) Protein kinase R 2-5 oligoadenylate synthetase/RNase L pathway 2 ,5 -Oligoadenylate Oasl2 synthetase-like 2 Oasl

2 ,5 -Oligoadenylate synthetase-like

Oas1b

2 ,5 -Oligoadenylate synthetase 1b

Oas1i

2 ,5 -Oligoadenylate synthetase 1

Oas1a

2 ,5 -Oligoadenylate synthetase 1a

Oas2

2 ,5 -Oligoadenylate synthetase 2

Rnase L

Ribonuclease L

Protein function

Induction/repression by MTH-68/H (fold)a,b

References

Cytokine (putative) Cytokine/type I IFN Cytokine/type III IFN (putative) Cytokine Transcription factor

80.3↑ 49.5↑ 23.0↑

Sadler and Williams (2008) Sadler and Williams (2008) Donnelly and Kotenko (2010)

15.2↑ 12.4↑

Sadler and Williams (2008) Sadler and Williams (2008)

Transcription factor

7.3↑

Sadler and Williams (2008)

Transcription factor Cytokine/type I IFN Cytokine/type I IFN Non-receptor tyrosine protein kinase Cytokine/type I IFN Type II IFN receptor Type II IFN receptor Type III IFN receptor Type III IFN receptor/IL10 receptor Type I IFN receptor Non-receptor tyrosine protein kinase Non-receptor tyrosine protein kinase Cytokine/type II IFN Type I IFN receptor

6.2↑ 4.0↑ 3.6↑ 3.4↑

Sadler and Williams (2008) Sadler and Williams (2008) Sadler and Williams (2008) Sadler and Williams (2008)

3.4↑ NSAc NSA NSA NSA

Sadler and Williams (2008) Sadler and Williams (2008) Donnelly and Kotenko (2010) Donnelly and Kotenko (2010) Sadler and Williams (2008)

NSA NSA

Sadler and Williams (2008) Sadler and Williams (2008)

NDd

Sadler and Williams (2008)

ND ND

Sadler and Williams (2008) Sadler and Williams (2008)

Ubiquitin-like protein E3 ubiquitin/ISG15 ligase

30.4↑ 28.7↑

Zhang and Zhang (2011) Zhang and Zhang (2011)

De-ISGylating protease E1 ubiquitin/ISG15 activating enzyme E3 ubiquitin/ISG15 ligase

27.8↑ 13.2↑

Zhang and Zhang (2011) Zhang and Zhang (2011)

6.2↑

Zhang and Zhang (2011)

De-ISGylating protease E2 ubiquitin/ISG15 conjugating enzyme E2 ubiquitin/ISG15 conjugating enzyme E2 ubiquitin/ISG15 conjugating enzyme

3.1↑ 2.4↑

Zhang and Zhang (2011) Sadler and Williams (2008)

2.3↑

Zhang and Zhang (2011)

2.3↑

Zhang and Zhang (2011)

Subunit of guanylate exchange factor Eif2␣ kinase

NSA

Sadler and Williams (2008)

NSA

Samuel (2001); Sadler and Williams (2008)

75.9↑

Sadler and Williams (2008)

43.5↑

Sadler and Williams (2008)

36.9↑

Sadler and Williams (2008)

13.6↑

Sadler and Williams (2008)

11.7↑

Sadler and Williams (2008)

5.7↑

Sadler and Williams (2008)

5.7↑

Bisbal and Silverman (2007)

Cytosolic viral RNA receptor/oligoadenylate synthetase Cytosolic viral RNA receptor/oligoadenylate synthetase Cytosolic viral RNA receptor/oligoadenylate synthetase Cytosolic viral RNA receptor/oligoadenylate synthetase Cytosolic viral RNA receptor/oligoadenylate synthetase Cytosolic viral RNA receptor/oligoadenylate synthetase ssRNA endonuclease

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A. Balogh et al. / Virus Research 185 (2014) 10–22

Table 2 (Continued) Gene/protein

Protein function

Induction/repression by MTH-68/H (fold)a,b

References

GTPase GTPase GTPase GTPase GTPase (putative) GTPase (putative)

75.8↑ 61.2↑ 51.0↑ 45.5↑ 33.7↑ 33.3↑

Vestal (2005) Vestal (2005) Vestal (2005) Akdis et al. (2011) Vestal (2005) Vestal (2005)

GTPase GTPase GTPase GTPase (putative) GTPase

32.1↑ 21.1↑ 15.7↑ 15.6↑ 14.8↑

Vestal (2005) Vestal (2005) Vestal (2005) Vestal (2005) Vestal (2005)

Viperin/antiviral protein

100.0↑

Seo et al. (2011)

Chemokine Translation regulator

98.7↑ 91.4↑

Raman et al. (2011) Schoggins et al. (2011)

Translation regulator

85.8↑

Schoggins et al. (2011)

Translation regulator

41.0↑

Schoggins et al. (2011)

Cytokine/type III IFN (putative) Cytokine Exonuclease RNA editing enzyme/antiviral protein RNA editing enzyme/antiviral protein

23.0↑

Donnelly and Kotenko (2010)

17.3↑ 7.0↑ 4.9↑

Akdis et al. (2011) Espert et al. (2003) Samuel (2001)

2.0↓

Samuel (2001)

Type II IFN effector pathways IFN␥ inducible protein 47 Ifi47 Gbp5 Guanylate-binding protein 5 Gbp1 guanylate-binding protein 1 Cxcl9 C–X–C motif ligand 9 Irgm Immunity-related GTPase M Oasl 2 -5 -Oligoadenylate synthetase-like Gbp4 Guanylate-binding protein 4 Gbp2 Guanylate-binding protein 2 Cxcl10 C–X–C motif ligand 10 Irf1 IFN regulatory factor 1 Tnfsf10 (TRAIL) TNF superfamily 10

Ras-like GTPase GTPase GTPase Chemokine Ras-like GTPase Cytosolic viral RNA receptor/OAS GTPase GTPase Chemokine Transcription factor Inflammatory cytokine

78.1↑ 75.8↑ 61.2↑ 48.9↑ 45.5↑ 43.5↑

Vestal (2005) Vestal (2005) Vestal (2005) Raman et al. (2011) Vestal (2005) Sadler and Williams (2008)

32.1↑ 21.1↑ 18.1↑ 17.5↑ 17.3↑

Il15 Ccl4 Igtp Nos2 Irf9 Adar Irf8

Cytokine Chemokine GTPase Nitric oxide synthase Transcription factor RNA editing enzyme Transcription factor

17.3↑ 16.8↑ 11.8↑ 6.9↑ 6.2↑ 4.9↑ 3.9↑

Vestal (2005) Vestal (2005) Raman et al. (2011) Savitsky et al. (2010) Humphreys and Halpern (2008) Akdis et al. (2011) Raman et al. (2011) Vestal (2005) Samuel (2001) Savitsky et al. (2010) Samuel (2001) Savitsky et al. (2010)

Guanylate-binding proteins Guanylate-binding protein 5 Gbp5 Gbp1 Guanylate-binding protein 1 Mx1 Myxovirus resistance protein 1 Irgm Immunity-related GTPase M 10801975 Similar to IFN-inducible GTPase 10819545 Similar to guanylate binding protein 1, IFN-inducible, 67 kDa Gbp4 Guanylate-binding protein 4 Gbp2 Guanylate-binding protein 2 Mx2 Myxovirus resistance protein 2 10801973 Similar to IFN-inducible GTPase Irg1 (p47GBP) p47 guanylate-binding protein Other type I IFN-regulated genes Radical S-adenosyl methionine Rsad2 domain containing 2 Cxcl11 C–X–C motif ligand 11 Ifit1 IFN-induced protein with tetratricopeptide repeats 1 Ifit2 IFN-induced protein with tetratricopeptide repeats 2 Ifit3 IFN-induced protein with tetratricopeptide repeats 3 10720237 Similar to IFN ␭2 Il15 Isg20 Adar

Interleukin 15 Exonuclease Adenosine deaminase, RNA specific

Adarb1

Adenosine deaminase, RNA specific b1

Interleukin 15 C–C motif ligand 4 IFN␥-induced GTPase Inducible nitric oxide synthase IFN regulatory factor 9 Adenosine deaminase, RNA specific IFN regulatory factor 8

For footnotes “a–d” see Table 1.

3.1.2. Toll-like receptor signaling Of the 12 members of the Toll-like receptor (TLR) family in rodents, three TLRs – TLR3, 7 and 9 – serve as transmembrane PRRs for viruses (Richards and Macdonald, 2011). Antiviral TLRs recognize viral nucleic acids. Activated TLR7 and 9 bind the adapter protein MyD88 (Fig. 1B) that in turn recruits the E3 ubiquitin ligase TRAF6 and protein kinases IRAK1 and 4. With the help of adapter proteins TAB1/2/3 the protein kinase TAK1 is activated and phosphorylates the IKK complex (IKK␣/IKK␤/NEMO). Phosphorylation of I␬B followed by its proteasomal degradation leads to nuclear translocation of the NF␬B transcription factors and induction of their target genes. TLR3 employes another adapter protein, TRIF, that serves as a platform for signal bifurcation: while TRAF6 feeds into the NF␬B pathway, TRAF3 couples TLR3 to IRF3/IRF7 signaling (see Fig. 1A).

Some of the genes coding for elements of TLR signaling were induced by MTH-68/H infection (e.g. Tlr3, Nfkb1, Nfkb2; Table 1). Interestingly, genes of I␬B isoforms that silence the NF␬B pathway (Nfkbia, Nfkbib, Nfkbid, Nfkbie, Nfkbiz) were also activated. Other genes of the pathway were not affected (Irak1/4, Traf3/6, etc.).

3.2. Interferon-stimulated pathways Genes of several type I IFNs (IFN␣1, IFN␣4, IFN␣11, IFN␤1) were strongly induced in MTH-68/H-infected PC12 cells (Table 1). Since their receptors, IFNAR1/IFNAR2 dimers are expressed ubiquitously (Samuel, 2001) autocrine/paracrine stimulation of type I IFN-signaling can be expected upon MTH-68/H infection of PC12 cell cultures.

A. Balogh et al. / Virus Research 185 (2014) 10–22

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Table 3 Expression of genes of cellular stress and apoptosis in MTH-68/H-infected PC12 cells. Gene/protein Intrinsic apoptosis pathway Apoptotic peptidase activating factor 1 Apaf1 Bid Casp7 Cyc1 Casp9 Casp3 Bad Bim Bcl-2 Bcl-xl Mcl-1 Bak Bax Tp53

BH3 interacting domain death agonist Caspase-7 Cytochrome c-1 Caspase-9 Caspase-3 Bcl-2-associated death promoter Bcl-2 interacting mediator of cell death B cell lymphoma-2 B cell lymphoma-extra large Myeloid cell leukemia sequence-1 Bcl-2 homologous antagonist killer Bcl-2 associated protein Tumor protein 53

Noxa/Pmaip1 Puma

PMA-induced protein 1 p53-upregulated modulator of apoptosis

Extrinsic apoptosis pathway Tumor necrosis factor (TNF superfamily, Tnf member 2) Tnfsf10 (TRAIL) TNF superfamily, member 10 (TNF-related apoptosis inducing ligand) Ripk2 Receptor-interacting serine-threonine kinase 2 Birc3 (cIAP2) Baculovirus IAP repeat-containing 3 (cellular inhibitor of apoptosis protein 2) Lta Lymphotoxin alpha (TNF superfamily, member 1) Birc2 (cIAP1) Baculovirus IAP repeat-containing 2 (cellular inhibitor of apoptosis protein 1 Tnfsf15 Tumor necrosis factor superfamily, member 15 Traf2 Tnf receptor-associated factor 2 Ripk3 Receptor-interacting serine-threonine kinase 3 Fas Fas (TNF receptor superfamily, member 6) Faslg Fas ligand (TNF superfamily, member 6) Ripk1 Receptor-interacting serine-threonine kinase 1 Tnf-r1 TNF receptor 1 Casp8 Caspase-8 Fadd Fas-associated death domain protein Tradd TNF receptor-associated death domain protein Trail-r1 TRAIL receptor 1 Trail-r2 TRAIL receptor 2 Endoplasmic reticulum stress pathway Activating transcription factor 3 Atf3 Casp4 Caspase-4 Casp12 Caspase-12 Ddit3(CHOP) DNA damage inducible transcript 3 (C/EBP homology protein) Traf2 TNF receptor-associated factor 2 Bip Binding Ig heavy chain protein Perk PKR-like endoplasmic reticulum kinase Ire1 Atf6 Xbp1 Nrf2 Jnk Atf4 Ask1

Inositol-requiring enzyme 1 Activating transcription factor 6 X-box binding protein 1 Nuclear factor (erythroid-derived 2)-related factor 2 c-Jun N-terminal kinase Activating transcription factor 4 Apoptosis stimulating kinase 1

Protein function

Induction/repression by MTH-68/H (fold)a,b

References

Scaffold protein component of apoptosome Pro-apoptotic Bcl2 family protein Effector caspase Apoptosis regulator Initiator caspase Effector caspase Pro-apoptotic Bcl2 family protein Pro-apoptotic Bcl2 family protein Anti-apoptotic Bcl2 family protein Anti-apoptotic Bcl2 family protein Anti-apoptotic Bcl2 family protein Pro-apoptotic Bcl2 family protein Pro-apoptotic Bcl2 family protein Transcription factor/tumor suppressor Pro-apoptotic Bcl2 family protein Pro-apoptotic Bcl2 family protein

4.6↑

Galluzzi et al. (2012)

2.6↑ 2.6↑ NSAc NSA NSA NSA NSA NSA NSA NSA NSA NSA NSA

Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012) Galluzzi et al. (2012)

NSA NDd

Galluzzi et al. (2012) Galluzzi et al. (2012)

Cytokine

35.4↑

Van Herreweghe et al. (2010)

Cytokine

17.3↑

Van Herreweghe et al. (2010)

Serine-threonine kinase

8.4↑

Van Herreweghe et al. (2010)

Adapter protein

6.1↑

Van Herreweghe et al. (2010)

Cytokine

6.0↑

Van Herreweghe et al. (2010)

Adapter protein

3.5↑

Van Herreweghe et al. (2010)

Cytokine

3.0↑

Van Herreweghe et al. (2010)

E3 ubiquitin ligase/adapter protein Serine-threonine kinase

2.6↑ 2.5↑

Van Herreweghe et al. (2010) Van Herreweghe et al. (2010)

Cytokine receptor Cytokine Serine-threonine kinase

2.3↑ 2.2↑ NSA

Galluzzi et al. (2012) Galluzzi et al. (2012) Van Herreweghe et al. (2010)

Cytokine receptor Initiator caspase Adapter protein Adapter protein

NSA NSA 2.0↓ 2.2↓

Van Herreweghe et al. (2010) Galluzzi et al. (2012) Van Herreweghe et al. (2010) Van Herreweghe et al. (2010)

Cytokine receptor Cytokine receptor

ND ND

Humphreys and Halpern (2008) Humphreys and Halpern (2008)

Transcription factor Initiator caspase Initiator caspase Transcription factor

40.1↑ 14.0↑ 11.8↑ 3.4↑

Jager et al. (2012) Jager et al. (2012) Jager et al. (2012) Jager et al. (2012)

Adapter protein ER chaperone UPR sensor/serine-threonine kinase UPR sensor/endoribonuclease UPR sensor/transcription factor Transcription factor Transcription factor

2.6↑ NSA NSA

Jager et al. (2012) Jager et al. (2012) Jager et al. (2012)

NSA NSA NSA NSA

Jager et al. (2012) Jager et al. (2012) Jager et al. (2012) Jager et al. (2012)

Serine-threonine kinase Transcription factor Serine-threonine kinase

NSA NSA ND

Jager et al. (2012) Jager et al. (2012) Van Herreweghe et al. (2010)

For footnotes “a–d” see Table 1.

3.2.1. Signaling to ISRE and GAS enhancer elements Ligand-bound IFNAR complexes activate the non-receptor tyrosine protein kinases JAK1 and TYK2 that in turn phosphorylate and activate components of the IFN-stimulated gene factor 3 (ISGF3)

complex consisting of the transcription factors STAT1, STAT2 and IRF9 (Fig. 2A). (The genes of all components of this ternary complex were induced in MTH-68/H-infected PC12 cells/Table 2.) ISGF3 is translocated to the nucleus and stimulates transcription of

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Fig. 1. Signaling pathways leading to type I IFN induction in virus-infected cells (based on Richards and Macdonald, 2011). (A) RIG-I-like receptor signaling. The family of RIGI-like receptors (RLR) is comprised of cytoplasmic RNA sensing proteins (RIG-I, MDA-5 and LGP2). Acting as cytoplasmic pattern recognition receptors (PRRs) they recognize viral RNA molecules. Upon virus infection RIG-I undergoes non-degradative polyubiquitination by the E3 ubiquitin ligase TRIM25, and thereby serves as a binding platform for the adapter protein IPS-1. IPS-1 is anchored to the outer mitochondrial membrane and activates the adapter proteins TRAF6 (that feeds into the NF␬B signaling pathway/see Fig. 1B/) and TRAF3. TRAF3 uses adapter proteins like TANK to bind and activate the IKK-related kinases TBK1 and IKK␧ that in turn phosphorylate the transcription factors IRF3 and IRF7 to induce the production of IFN␣/␤. (B) Toll-like receptor signaling. Three members of the Toll-like receptor (TLR) family are involved in the antiviral response of cells: TLR3, TLR7 and TLR9. They act as transmembrane PRRs recognizing viral nucleic acid components. Upon ligand binding, TLR7 and TLR9 recruit the cytoplasmic adapter protein MyD88 that forms a signaling complex with TRAF6 and with the serine/threonine specific protein kinases IRAK1 and IRAK4. With the help of additional adapter proteins (TAB1/2/3) the protein kinase TAK1 is activated that, in turn, phosphorylates and stimulates the I␬B kinases IKK␣ and IKK␤. Binding of a regulatory subunit, NEMO, creates the IKK complex that phosphorylates I␬B proteins sequestering NF␬B in the cytoplasm. Phosphorylation of I␬Bs leads to their ubiquitination and proteasomal degradation, NF␬B is released, translocated to the nucleus and induces the transcription of target genes including genes of IFN␤ and inflammatory cytokines. The activated TLR3 binds the adapter protein TRIF that serves as a platform for a two-armed signaling pathway. TRIF associates with TRAF6 and the protein kinase RIP1 that couple the TLR3 receptor to the NF␬B signaling pathway. TRIF is also able to bind TRAF3 that links TLR3 to the pathway mediated by the IRF3/7 transcription factors (see above). (For abbreviations of signaling proteins see Table 1.)

hundreds of IFN-stimulated genes (ISGs) carrying IFN-stimulated response element (ISRE) enhancers (Sadler and Williams, 2008; Samuel, 2001). Remarkably, a putative type III IFN gene (designated #10720237 in Table 2) coding for a protein similar to IFN␭2 was also strongly induced by MTH-68/H. Since the pathways leading to IFN␭ production and its biological effects are similar to those of type I IFNs (Donnelly and Kotenko, 2010), it may support the effects of IFN␣ and ␤. Interestingly, although the type II IFN, IFN␥, was not induced in MTH-68/H-infected PC12 cells, its target genes (Table 2) appeared to be stimulated by the virus. A possible explanation for this observation may be that STAT1 homodimers – strongly induced by MTH-68/H infection and activated through type I IFN receptors – translocate to the nucleus and activate genes with IFN␥-activated sequence (GAS) enhancers.

Since a large number of type I and type II IFN-regulated genes were induced by MTH-68/H, both IFN pathways appear to be functional in PC12 cells. 3.2.2. Type I IFN effector pathways Hundreds of genes are induced in cells treated with type I IFNs (Sadler and Williams, 2008; Schoggins et al., 2011). While the main role of proteins encoded by these ISGs is to provide an innate antiviral response, a wide range of other cellular effects (e.g. stimulation of apoptosis, modulation of RNA and protein metabolism, remodeling of the cytoskeleton, etc.) is evoked by these gene products. Four ISG effector pathways are especially important in the innate immune response of cells to virus infection: the ISG15, protein kinase R (PKR), 2 ,5 -oligoadenylate synthetase (OAS)/RNase L and Mx pathway (Samuel, 2001; Sadler and Williams, 2008).

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Fig. 2. IFN signaling (based on Sadler and Williams, 2008). (A) Induction of IFN-stimulated genes (ISGs) by IFN␣ and IFN␤ Type I IFNs bind to the heterodimeric receptor IFNAR1/IFNAR2 activating the non-receptor tyrosine protein kinases JAK1 and TYK2. Phosphorylation of the receptor by these protein kinases attracts STAT1 and STAT2 proteins that, upon phosphorylation, form a ternary complex STAT1/STAT2/IRF9 (ISGF3 complex). ISGF3 translocates to the nucleus and induces ISGs containing an ISRE enhancer element. Of the 380 ISGs the genes of four classes of proteins are particularly important and well characterized: ISG15, PKR, OAS (its product activates RNase L) and Mx. (ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element). (B) Stimulation of IFN␥-regulated genes by IFN␣ and IFN␤. IFN␣/␤-activated STAT1 molecules are able to form homodimers as well that are key mediators of the type II IFN, IFN␥. IFN␣/␤ signaling may thus be coupled to a set of genes containing the GAS enhancer element thereby leading to the induction of IFN␥-regulated genes. (This possible cross-talk is indicated by the dotted line; GAS, IFN␥-activated sequence.)

The Isg15 gene (Zhang and Zhang, 2011) codes for an ubiquitinlike protein that can be linked to a large number of target proteins including proteins of antiviral responses (e.g. RIG-I, IRF3, JAK1, STAT1, MxA, PKR, RNase L, etc.). Importantly, many of the ISG15 targets are components of stress signaling. ISGylation regulates the activity of target proteins. Enzymes catalyzing protein ISGylation are similar to ubiquitinating enzymes; all of them are induced by IFN␣/␤ (Sadler and Williams, 2008). MTH-68/H infection of PC12 cells up-regulated the Isg15 gene and several genes involved in its metabolism (Uba7, Ube2L6, Herc6, Trim25). Interestingly, genes coding for de-ISGylating proteases (Usp18, Usp49) were also induced by MTH-68/H (Table 2). PKR is an RNA-dependent protein kinase (Sadler and Williams, 2008; Samuel, 2001): it binds viral RNA molecules, undergoes activating autophosphorylation and caspase-dependent proteolytic cleavage and phosphorylates target proteins. Its most important substrate is eIF2␣, a translational initiation factor; phosphorylation of eIF2␣ leads to the inhibition of translation. PKR is up-regulated by type I IFNs in virus-infected cells. The shut-down of protein synthesis is mainly responsible for its antiviral and antiproliferative effect. PKR is strongly expressed in PC12 cells and is proteolytically activated by various pro-apoptotic stimuli (Pap and Szeberenyi, 2008).

One of the most strongly stimulated pathways in MTH-68/Hinfected cells was the OAS/RNase L pathway (Table 2). OAS enzymes (Bisbal and Silverman, 2007; Sadler and Williams, 2008; Samuel, 2001) are activated by dsRNA and induced by type I and II IFNs. They synthesize 5 -triphosphorylated oligoadenylates with 2 -5 phosphodiester bonds (2-5A). The only target of 2-5A is the endoribonuclease RNase L: upon activation by 2-5A it cleaves both viral and cellular RNA molecules. Since several Oas genes and the Rnase L gene were strongly induced by MTH-68/H, the OAS/RNase L pathway may be an important contributor to the oncolytic effect of NDV. Several members of the dynamin superfamily of guanylatebinding proteins (GBPs) are induced by type I/II IFNs (Sadler and Williams, 2008; Samuel, 2001; Vestal, 2005). The best characterized GBPs are the Mx proteins (Mx1 and Mx2 in rodents). The Mx genes are induced by IFN␣/␤, but not by IFN␥, neither are they activated directly by virus-infection; they serve as markers of type I IFN-responsiveness (Vestal, 2005). Mx1 is a powerful antiviral protein: it is alone sufficient to block virus replication (Mx2 does not show antiviral activity). The antiviral role of other GBPs, although the genes of some of them were strongly induced by MTH-68/H, has not been proven.

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Many other ISGs were induced by MTH-68/H in PC12 cells (some of them are listed in Table 2). The Rsad2 gene codes for the antiviral protein viperin (Seo et al., 2011). Viperin is induced in most cell types by IFN␣/␤, and has a broad antiviral spectrum. Rsad2 displayed the highest level of induction by MTH-68/H. Chemokines are secreted proteins that promote directional migration of cells (Raman et al., 2011). They regulate a wide range of processes (innate and acquired immunity, embryogenesis, angiogenesis, tumorigenesis, etc.). Many chemokines and their receptors are expressed in PC12 cells. The Cxcl11 gene was strongly induced by MTH-68/H, presumably via the IFN␣/␤ pathway. Other chemokine genes (Cxcl9, Cxcl10, Ccl4) are activated through IFN␥ signaling. Although the receptors for these chemokines (CXCR3 and CCR1) are also expressed in PC12 cells, their role in direct cytotoxicity of MTH-68/H is not clear. Members of the Ifit gene family are strong antiviral ISGs: their products inhibit translation in virus-infected cells (Schoggins et al., 2011). The Ifit1, Ifit2 and Ifit3 genes were all strongly induced in MTH-68/H-infected PC12 cells. Because of their effect on protein synthesis they may trigger death signaling as well. 3.2.3. Type II IFN effector pathways IFN␥ is an immune IFN secreted by certain cells of the immune system (Samuel, 2001). Although its gene was not up-regulated by MTH-68/H (see Section 3.2.1), several genes regulated by IFN␥ via GAS enhancer elements were found strongly induced (Table 2), presumably through a crosstalk from IFN␣/␤ signaling (see Fig. 2). Of the protein products of genes induced by MTH-68/H via IFN␥ signaling GTPases (GBPs, IFI47, IRGM, IGTP) may generate antiviral resistance, secreted ligands (chemokines CXCL9, CCL4, cytokines TRAIL, IL-15) may trigger diverse cellular responses, IRF1 (and other IFN-regulatory factors) may activate further target genes. 3.3. Pathways of cellular stress and apoptosis MTH-68/H-infected PC12 cells die by apoptosis (Fabian et al., 2007). Many genes involved in the activation of the receptormediated and mitochondrial pathways of apoptosis and various forms of cellular stress (Figs. 3 and 4) were affected in MTH-68/Hinfected PC12 cells (Table 3). 3.3.1. The intrinsic (mitochondrial) apoptosis pathway Stress signals acting intracellularly (DNA damage, toxic compounds etc.) activate the mitochondrial pathway of apoptosis (Galluzzi et al., 2012; Van Herreweghe et al., 2010; Fig. 3A). Bcl-2 protein family members regulate the release of apoptosis-inducing proteins (e.g. cytochrome c) through the outer mitochondrial membrane. Cytochrome c forms a complex with caspase-9 and the structural protein Apaf-1. The apoptosome proteolytically activates effector caspases (caspase-3, -6 and -7) that execute apoptosis by cleaving a large number of caspase substrate proteins. The intrinsic pathway of apoptosis was only mildly affected by MTH-68/H in PC12 cells at the level of the transcriptome (Table 3): only Bid, Apaf-1 and Casp7 were slightly induced. This is not surprising, since the function of the key transcriptional regulator of this pathway, the tumor suppressor p53 protein, is not essential for MTH-68/H cytotoxicity (Fabian et al., 2007). 3.3.2. The extrinsic (receptor-mediated) apoptosis pathway MTH-68/H infection of PC12 cells leads to the induction of genes coding for inflammatory cytokines TNF, TRAIL and FasL (Humphreys and Halpern, 2008; Van Herreweghe et al., 2010; Table 3). These death ligands, upon binding to their receptors, stimulate several cellular stress mechanisms including the intrinsic pathway of apoptosis (Fig. 3B).

Of these ligands TRAIL appears to be the most important autocrine/paracrine mediator of virus cytotoxicity. Although the Tnf gene was very strongly induced, TNF has only a weak apoptotic effect in PC12 cells (unpublished observations). FasL and its receptor were only weakly induced by (Table 3). In contrast, Tnfsf10 (the gene coding for TRAIL) was strongly up-regulated by the virus and TRAIL is a powerful stimulator of p53-independent death signaling. Its mediator role in NDV-induced apoptosis was suggested earlier (Elankumaran et al., 2006) and may be an important contributor to MTH-68/H cytotoxicity. 3.3.3. Endoplasmic reticulum stress MTH-68/H replicates in PC12 cells and triggers events of ER stress (activation of caspase-12, phosphorylation of PERK and eIF2␣) that are implicated in cell death (Fabian et al., 2007). ER stress is characterized by the accumulation of unfolded proteins in the lumen of ER leading to a chain of processes known as unfolded protein response (UPR; Jager et al., 2012). Misfolded proteins stimulate the UPR sensors PERK, IRE1, ATF6 and caspase-12/4 that activate pathways of survival (by trying to restore ER homeostasis through the induction of UPR target genes) and cell death (via caspase-dependent apoptosis; see Fig. 4 for details). MTH-68/H strongly induced several genes (Casp4, Casp12, Ddit3, Atf3) that code for proteins of the pro-apoptotic solution of ER stress (Table 3). Induction of these genes and stimulation of downstream pathways by their products may be essential for the oncolytic potential of MTH-68/H. 4. Discussion The aim of the present study was to analyze the effects of MTH68/H, an oncolytic NDV strain, on gene expression profiles in the PC12 cell line. This cell line provides a useful model system to identify genes involved in the antiviral response and in the ultimate cell demise of virus-infected cells. Analysis of gene expression changes identified signaling pathways that may contribute to the susceptibility/resistance of tumor cells toward NDV. Limited information is available on genome-wide gene expression changes in NDV-infected cells. Munir et al. (2005) studied gene expression in primary chicken embryo cells infected with a velogenic NDV strain using a 2950-element cDNA chip and found 22 and 33 genes up- and down-regulated, respectively. The most detailed study so far was conducted by Krishnamurthy et al. (2006). These authors used a whole-genome cDNA microarray and compared the transcriptomes of normal human skin fibroblasts and the HT-1080 human fibrosarcoma cell line infected with NDV. Several genes were differentially regulated in the two cell lines. 4.1. Hundreds of genes were affected in MTH-68/H-infected PC12 cells Infection of PC12 cells with MTH-68/H at an MOI corresponding to the IC50 for 12 h triggered substantial transcriptional alterations: 729 and 612 genes were up- and down-regulated, respectively (see Supplementary Tables S1 and S2). Changes were especially dramatic in the case of up-regulated genes (Supplementary Table S1): more than 50 genes were induced 50-fold or higher and another 70 genes were expressed 10- to 50-fold of control cells. Considering the fact that cells try to generate a strong antiviral response while the virus replicates and triggers apoptotic signaling, such dramatic gene expression changes could be anticipated. Supplementary Tables S1 and S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.03.003. The up- and down-regulated genes were classified into 176 and 146 overlapping clusters, respectively. Several up-regulated gene

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Fig. 3. Pathways of cellular stress and apoptosis (based on Van Herreweghe et al., 2010). (A) Intrinsic apoptosis pathway. Growth factor withdrawal, DNA damage and other cellular stressors stimulate the intrinsic (mitochondrial) pathway of programmed cell death. The BH3-only members of the Bcl-2 family of apoptosis regulating proteins (Bid, Bad, Bim, Noxa, Puma) become induced/activated (Noxa and Puma are directly induced by the DNA damage-stimulated p53 protein). BH3-only proteins activate the multi-domain pro-apoptotic members of the Bcl-2 family (Bak and Bax) by displacing anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL, Mcl-1). Bak and Bax form pores in the outer mitochondrial membrane and pro-apoptotic proteins, including cytochrome c, are released to the cytosol from the intermembrane space. Cytochrome c forms apoptosomes with the initiator caspase caspase-9 and the scaffold protein Apaf-1. Caspase-9 cleaves and activates effector pro-caspases and the effector caspases-3/6/7 execute apoptosis by cutting a number of caspase substrates. (B) Extrinsic apoptosis pathway. Apoptosis can be induced by death ligands (TNF, FasL, TRAIL) binding to their cognate death receptors (TNFR, Fas, TRAIL-RI/II; receptor-mediated pathway). Death receptors oligomerize upon ligand binding, become activated and bind specific adapter proteins (TRADD, FADD, TRAF2, cIAP) forming a death-inducing signaling complex (DISC). Binding of initiator pro-caspases-8/10 to the receptor leads to their auto-proteolytic activation. Caspase-8/10 then cleave and activate effector pro-caspases-3/6/7. Caspase-8 is also able to cut and activate the pro-apoptotic Bid protein. Besides this apoptotic mechanism the cellular stress pathways mediated by the stress kinase JNK and the transcription factor NF␬B are also regulated by death receptors (for abbreviations of signaling proteins see Table 3).

clusters are involved in the innate immune response, inflammation and death signaling of the infected cells. Genes of cell cycle regulation and cellular metabolism were typically down-regulated. The effect of the virus on a few signaling mechanisms will be discussed here. 4.2. MTH-68/H evoked a strong IFN response in PC12 cells The virotherapeutic potential of NDV is based on its selective cytotoxicity toward tumor cells. The basis for this tumor

selectivity, however, is currently not clear. One possible explanation was suggested by Lorence et al. (2007): tumor cells unable to generate an IFN response upon NDV infection are killed by the virus while normal cells are protected by their strong IFN-mediated antiviral response. Detailed analysis of HT1080 human fibrosarcoma cells indicated that, while their NDV-stimulated IFN secretion was normal, a defect in IFN signaling made them susceptible to virus cytotoxicity (Krishnamurthy et al., 2006). This observation strongly supports the hypothesis described above.

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Fig. 4. Endoplasmic reticulum stress response (based on Jager et al., 2012). Accumulation of misfolded proteins in the ER lumen may lead to unfolded protein response (UPR) or apoptotic cell death. Unfolded proteins bind and sequester the ER lumen chaperone BiP that leads to the activation of UPR sensor proteins PERK, IRE1 and ATF6. PERK phosphorylates the translation initiation factor eIF2␣ that leads to a general inhibition of protein synthesis, but an enhanced translation of the transcription factor ATF4. ATF4 stimulates the expression of target genes including that of the pro-apoptotic transcription factor CHOP. PERK phosphorylates the pro-survival transcription factor NRF2 as well that translocates to the nucleus and induces proteins that maintain redox homeostasis. IRE1 stimulates the accumulation of the transcription factor XBP1 and expression of its target genes coding for ER chaperones. Prolonged activation of IRE1 leads to the stimulation of the JNK pathway. Transmembrane transcription factor ATF6 is cleaved, released and translocated to the nucleus and regulates several UPR target genes. ER stress also triggers the proteolytic activation of ER-bound procaspase-12/4 that, through downstream caspases, induce apoptotic cell death. (For abbreviations of signaling proteins see Table 3.)

IFNs, a family of cytokines, have crucial roles in antiviral and antitumor responses (Sadler and Williams, 2008; Samuel, 2001). Type I (or viral) IFNs, IFN␣ and IFN␤, are produced by many cells upon virus infection. The type II (or immune) IFN, IFN␥, is secreted by certain immune cells and is involved in the long-term control of viral infection. Type III IFNs (IFN␭) are induced by many viruses and their biological functions are similar to those of IFN␣/␤ (Donnelly and Kotenko, 2010). The role of the IFN pathway defects in NDV-susceptibility has been challenged recently by several laboratories. Primary human melanoma cell cultures (Lazar et al., 2010), and the A549 human non-small-cell lung cancer cell line (Mansour et al., 2011) were found to be killed by NDV despite having intact IFN signaling. Type I IFN signaling appears to play the main role in the acute response of PC12 cells to MTH-68/H infection. Several genes coding for IFN␣ and ␤ isoforms were strongly induced after 12 h of infection by MTH-68/H in PC12 cells (gene #10869879 coding for a precursor to IFN␣6 and IFN␣8, Ifna1, Ifna4, Ifna11, Ifnb1, Ifnb2, see Table 1). The fact that a large number of type I IFN effector genes were also activated (Table 2) provided strong evidence that the increase of transcription led to elevated levels of IFN␣ and ␤ proteins and to their secretion. Importantly, several elements of RLR signaling (e.g. RIG-I, MDA5, LGP2, DDX60, TRIM25, IRF7) were induced at the RNA level as well, some of them presumably through IFN signaling. This important innate immune response pathway was thus – besides being directly stimulated by viral components – further amplified by the induction of several of its key elements by NDV. The genes coding for several components of the TLR pathway (e.g. Tlr3, MyD88, NF␬B genes) were only moderately induced by MTH-68/H. We thus may conclude that the main signal

transduction pathway that mediates the stimulation of type I IFN production by MTH-68/H in PC12 cells is the RLR pathway. IFN␣ and IFN␤ secreted by MTH-68/H-infected PC12 cells may act by autocrine and paracrine mechanisms in the cell cultures. Besides stimulating the type I IFN pathway, several elements of this mechanism were also induced at the transcriptional level (e.g. type I IFN genes/Table 1/, Stat1, Stat2, Irf9, Table 2). More than 380 genes, termed ISGs, are induced by type I IFNs (Schoggins et al., 2011). Several elements of the major type I IFN effector pathways (ISG15, guanylate-binding proteins, 2-5 OAS/RNase L) were induced by MTH-68/H infection (Table 2). Some of these pathways may trigger pleiotropic effects: the ubiquitinlike ISG15 protein, for example, may regulate more than 150 target proteins involved in cellular antiviral or stress responses (Sadler and Williams, 2008). Furthermore, the ISG15 pathway provides another example for signaling complexity: both ISGylating (e.g. Uba7, Ube2E1, Herc6) and de-ISGylating enzymes (e.g. Usp18, Usp49) were induced by MTH-68/H infection. Guanylate binding proteins are indicators of IFN-responsiveness (Vestal, 2005): strong induction of some of them by MTH-68/H (e.g. Mx1, Mx2) clearly indicates that PC12 cells are both able to secrete type I IFNs and to respond to these cytokines. Some of the GBPs are antiviral (e.g. GBP5, Mx1), while others inhibit cell proliferation (e.g. GBP1). Induction of genes of the OAS/RNase L pathway (members of the Oas gene family, Rnase L; Table 2) may contribute to both the antiviral and apoptotic responses to MTH-68/H depending on the conditions. The PKR pathway was not induced by MTH-68/H at the transcriptional level (but was found to be activated at protein level/Fabian et al., 2007/). Many other type I IFN effector genes were strongly induced (see Table 2). Some of them code for powerful antiviral proteins (e.g.

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Rsad2, Ifit genes, Isg20, Adar), others for paracrine modulators of immune cells (e.g. Cxcl11, Cd274, gene #10720237), pro-apoptotic proteins (e.g. Casp4, Casp12, Casp7), inhibitors of the cell cycle (Samd9l, Slfn5). IFN␥ target genes were also induced by MTH-68/H infection of PC12 cells (see Table 2). Some of the IFN␥-regulated genes are also stimulated through IFN␣/␤ signal transduction (e.g. Stat1, Gbp2), others are preferentially induced by IFN␥. Some of these genes may be strongly implicated in MTH-68/H cytotoxicity (e.g. Irgm, Irf1, Tnfsf10). It thus appears that PC12 cells are able to produce a robust IFN response to infection by the MTH-68/H strain, and this response does not prevent NDV replication and death of the cells. 4.3. MTH-68/H shifts the pattern of gene expression toward a cytostatic and cytocidal response MTH-68/H infection of PC12 cells affected the expression of a number of cell cycle regulatory genes, both stimulators (e.g. Figf, Fgf1l, EphB2, Pole2, Cdc26) and inhibitors (e.g. Cnksr3, Btg2, Klf10; see Supplementary Tables S1 and S2). Genes were up- and down-regulated in both categories, but the overall balance of gene expression changes favors cell cycle arrest. A large body of experimental data supports the notion that NDV kills tumor cells by apoptosis (Alabsi et al., 2011, 2012; Ali et al., 2011; Fabian et al., 2007; Zulkifli et al., 2009). The mechanism is, however, still a matter of controversy. Data from several laboratories indicate that both the extrinsic and intrinsic pathways of apoptosis are involved in NDV oncolysis (Bian et al., 2011; Ravindra et al., 2009), while other researchers suggest that cell killing is mostly mediated by the mitochondrial pathway of apoptosis (Elankumaran et al., 2010; Meng et al., 2012; Molouki et al., 2010). The significance of virus replication and the involvement of ER stress have also been suggested (Fabian et al., 2007). The solution of the debate is at present impossible: various isolates of NDV with different virulence and a number of different cell lines were used in these studies; the results are therefore not directly comparable. Analysis of the transcriptome of PC12 cells conducted in the present study indicated that the expression of several genes in the extrinsic, intrinsic and ER stress-induced apoptosis pathways was affected during the early phase of MTH-68/H infection. Based purely on the gene expression data the death receptor and ER stress-mediated mechanisms appear to be particularly important (see Table 3). 4.4. Why are PC12 cells killed by MTH-68/H? Based on the microarray data presented in this paper MTH68/H appeared to evoke a pleiotropic response in PC12 cells: a wide range of signaling pathways with different phenotypic outcomes were affected. Most of these mechanisms are able to act both ways: they simultaneously activate antiviral and pro-apoptotic responses. Type I and III IFNs, for example, are mediators of antiviral (thus pro-survival) and pro-apoptotic pathways as well (Donnelly and Kotenko, 2010; Richards and Macdonald, 2011; Samuel, 2001). Some of the ISGs strongly up-regulated by MTH-68/H (see Table 2) like Mx1 (Samuel, 2001), Rsad2 (Seo et al., 2011) Ifit family of genes (Schoggins et al., 2011) code for antiviral proteins. Others may trigger cell death. A typical example of a mechanism acting as a double-edged sword is the OAS/RNase L pathway (Bisbal and Silverman, 2007; Sadler and Williams, 2008; Samuel, 2001). Several members of the Oas gene family were strongly up-regulated by MTH-68/H infection in PC12 cells (Oasl, Oasl2, Oas1a, Oas1b, Oas1i; Table 2). 2-5A oligonucleotides synthesized by OAS activate endonuclease RNase L (its gene is also induced by MTH-68/H) that degrades both viral and cellular RNAs (Sadler and Williams, 2008; Samuel, 2001). At low 2-5A concentrations the antiviral effect

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dominates, but high concentrations of 2-5A are cytotoxic by triggering cleavage of rRNA molecules (Bisbal and Silverman, 2007). A similar phenomenon can be observed during the UPR: depending on the intensity of ER stress the fate of the cell can be decided by pro-survival or pro-apoptotic mechanisms. At mild ER stress conditions the IRE1 UPR sensor and the transcription factors ATF4, XBP1 and NRF2 up-regulate genes coding for ER chaperones thereby increasing the folding capacity of the ER. Under severe ER stress conditions, however, the signaling switches from an adaptive to a pro-apoptotic response mainly due to the induction of the strongly apoptotic transcription factor CHOP (Jager et al., 2012). As shown in Table 3, MTH-68/H infection in PC12 cells affected only a few ER stress genes (Atf3/a target gene of CHOP/, Casp4, Casp12 and Ddit3/that codes for CHOP/), but the products of all these genes “favour” cell killing over survival. Another example of life/death decision in MTH-68/H-infected PC12 cells is the case of death ligands. The genes of both TNF and TRAIL were strongly up-regulated by the virus (see Table 3). While the NF␬B-mediated pro-survival effects of TNF overcome its weak apoptotic action (Van Herreweghe et al., 2010), TRAIL may act as a strong activator of cell death (Humphreys and Halpern, 2008). We may thus conclude that the susceptibility of individual tumor cells to NDV is determined by the delicate balance of antiviral and pro-apoptotic mechanisms activated by infection. Key proteins of these signaling pathways may serve as useful NDV sensitivity markers. 4.5. The in vivo oncolytic effect of MTH-68/H may be amplified by the release and paracrine effect of various cytokines and chemokines Most of the genes up- or down-regulated by MTH-68/H in PC12 cells code for proteins located inside the cell and thus affect the fate of individual cells infected by the virus. Other genes express secreted proteins that may act by autocrine or paracrine signaling targeting the secretory cell or neighboring tumor cells, respectively (e.g. type I and III IFNs, TNF, TRAIL), provided that they express the cognate receptors of these ligands. A third group of genes with altered expression in MTH-68/H-infected PC12 cells cannot affect the behavior of these cells in culture: the encoded proteins may influence the function of other cell types under in vivo conditions. Interleukins induced by MTH-68/H (IL6, IL15) may enhance antitumor immunity (Akdis et al., 2011). Several chemokines strongly up-regulated by the virus at the RNA level may act as chemoattractants for immune cells (CCL4, CXCL9, CXCL10, CXCL11) or as angiostatic agents (CXCL9, CXCL10, CXCL11; Raman et al., 2011). Down-regulation of certain growth factors may also inhibit angiogenesis (Figf/VEGFD; Achen et al., 1998) or tumor invasion (Fgf11; Itoh and Ornitz, 2008). Such gene expression changes may thus enhance the in vivo oncolytic power of NDV without directly affecting the viability of the infected cell. The present study revealed that infection of PC12 cells with the attenuated NDV strain MTH-68/H triggered fundamental gene expression changes. Information obtained with this rodent cell line can be used in studies to be conducted on MTH-68/H infected human tumor cells. Detailed analysis of the role of affected key signaling proteins will contribute to the understanding of the oncolytic mechanisms of NDV and factors influencing tumor cell susceptibility to this candidate virotherapeutic agent. Acknowledgements The technical assistance of Ms. Zita Árvai, Ms. Ibolya Koloszár and Ms. Mónika Vecsernyés is greatly acknowledged by the authors. This work was supported by Science, Please Research Team on Innovation (grant number SROP – 4.2.2./08/1/2008-2011).

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Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain.

Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV...
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