Modulation of Host Cell Gene Expression During Onset of the Late Phase of an Adenovirus Infection Is Focused on Growth Inhibition and Cell Architecture

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Virology 343 (2005) 236 – 245 www.elsevier.com/locate/yviro

Modulation of host cell gene expression during onset of the late phase of an adenovirus infection is focused on growth inhibition and cell architecture

Fredrik Granberg a, Catharina Svensson b, Ulf Pettersson a, Hongxing Zhao a,*

a Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden b Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, Box 582, S-741 23 Uppsala, Sweden

Received 22 June 2005; returned to author for revision 1 August 2005; accepted 17 August 2005 Available online 16 September 2005

Abstract

Microarray analysis of host cell gene expression during an adenovirus type 2 infection showed that the number of regulated genes, as well as the magnitude of change, was increased as the infection proceeded into the late phase. In contrast to the early phase of infection when the majority of differentially expressed genes were upregulated, expression of most of the regulated genes (82 out of 112) declined during the late phase. In particular, numerous TGF-h inducible genes and several TGF-h-independent growth-arrest-inducing genes were targeted. Of the 30 genes upregulated more than 2-fold at 20 h post-infection, nearly two-thirds of encoded proteins are involved in cell metabolism. The data indicate that adenovirus primarily targets cellular genes involved in antiviral defense, cell growth arrest and apoptosis, as well as cell metabolism, to ensure sufficient production of viral progeny. D 2005 Elsevier Inc. All rights reserved.

Keywords: Adenovirus; Microarray; Late phase; Gene expression

Introduction to obtain efficient expression from the adenovirus E2 promoter (Kovesdi et al., 1986). In addition, the E4 orf6/7 further To obtain efficient production of progeny virus, human activates E2 expression by stabilizing the interaction of E2F to adenovirus must ensure high expression of its genes, induce the duplicated E2F binding sites in the E2 promoter used favorable conditions for viral DNA replication and counteract specifically during the early phase of infection (Bagchi et al., the host cell antiviral defense. Parts of these goals are achieved 1990; Neill et al., 1990). E1A also targets the transcriptional by modifying the transcriptional machinery of the infected cell. coactivators p300/CBP, which have been implicated in multiple Activation of the adenovirus early genes is primarily achieved cellular processes by interacting with a growing list of through interaction between the conserved region 3 (CR3) of transcription factors (Arany et al., 1995; Bannister and the immediate adenovirus E1A protein and the Sur2 subunit of Kouzarides, 1995). As coactivators, p300/CBP act not only the Mediator complex (Wang and Berk, 2002), probably as transcriptional integrators creating architectural scaffolds for increasing the efficiency of RNA polymerase II recruitment transcription complex assembly, but also as chromatin modi- to viral promoters. Induction of the S-phase, on the other hand, fiers through their intrinsic acetyl transferase activities (Chan is mainly ascribed to the ability of E1A to bind members of and La Thangue, 2001). Although the ability of E1A to bind retinoblastoma tumor suppressor (pRb) family. By binding to and sequester p300/CBP has been described as one of the pRb, E1A will alleviate the inhibitory pRb–E2F interaction, major explanations for transcriptional repression by E1A, thereby activating expression of E2F-dependent genes (Cobri- mainly leading to the induction of cell proliferation (Arany et nik, 1996). Although this has been proposed to cause a al., 1995; Bannister and Kouzarides, 1995), E1A may also significant deregulation of host cell E2F-dependent genes redirect p300/CBP to activate specific target genes (Fax et al., (Zhao et al., 2003), the transcription factor E2F is also required 2000). Additional possibilities for E1A to deregulate cellular transcription are given by the ability of E1A to bind to p400/ * Corresponding author. Fax: +46 18 471 4808. TRRAP containing chromatin-remodeling complexes (Deleu et E-mail address: [email protected] (H. Zhao). al., 2001; Fuchs et al., 2001; Lang and Hearing, 2003;

Nikiforov et al., 2002) and to the corepressor C-terminal are preferentially translated after reaching the cytoplasm. Binding Protein (CtBP) (Chinnadurai, 2004; Johansson et al., Expression of the L4-100 kDa protein from the major late 2005). Thus, the ability of E1A to target different cellular transcription unit has been implicated in regulation of viral proteins involved in transcriptional regulation offers a variety gene expression and turning off expression of cellular genes of possibilities to modulate the host cell growth controlling (Farley et al., 2004; Hayes et al., 1990; Cuesta et al., 2000). activities. The knowledge about the regulation of host cell gene Furthermore, adenovirus early genes also target the cellular expression at the transcriptional level during adenovirus p53 tumor suppressor protein which blocks cell cycle infection is limited. Microarray technology has proved to be progression. In addition to the direct binding to p53, thereby a powerful tool to monitor changes in the expression of blocking p53-dependent transcriptional activation (Sarnow et thousands of host cell genes in response to infectious al., 1982; Yew et al., 1994), E1B-55 kDa protein also forms pathogens. Our previous studies have focused on the host cell complex with E4 open reading frame 6 (E4orf6) protein and gene expression during the early phase of adenovirus infection. mediates the ubiquitylation and degradation of p53 (Querido et We showed that adenovirus modulates expression of a limited al., 2001; Steegenga et al., 1998). set of cellular genes at 6 h post-infection of HeLa cells. Here, As described, most of the direct effects on host cell gene we have extended our study to the late phase of the infection expression during the early phase are due to E1A. Since the and describe virus-induced changes in gene expression at 10 p300/CBP coactivators act on transcription factors controlling and 20 h post-infection. genes involved in antiviral defense, it is conceivable that E1A directly deregulates these genes. However, the ability of Results adenovirus to interfere with the host antiviral response also involves the capacity of adenovirus E3 proteins to interrupt Augmented regulation of host gene expression during antigen presentation and pro-inflammatory signaling (Wold et progression of an adenovirus infection al., 1994). The E3-gp19K prevents exposure of viral peptides on the cell surface by inhibiting the transport of class I major Using cDNA microarray analysis, we have previously histocompatibility complex from the ER to the plasma studied adenovirus type 2-induced modification of host cell membrane as well as inhibiting the loading of peptide by gene expression at early time (6 h) of infection (Zhao et al., tapasin (Bennett et al., 1999; Burgert et al., 1987; Wold et al., 2003). In this study, we measured cellular gene expression as 1999). The E3-10.4 K and 14.5 K (RIDa/h) complex prevents the virus infection proceeded into the late phase. Quantification tumor necrosis factor alpha (TNFa) and Fas ligand-induced of viral gene expression using the adenovirus amplicons cell death by destroying the death domain-containing receptors present on the microarray showed that adenovirus early genes through internalization and, subsequently, degradation in the were expressed to high levels at 10 h post-infection, whereas lysosome. In addition, the E3-10.4 K/14.5 K complex also expression of the late genes, L1 and L3, was still very low (data blocks the activation of NF-nB by preventing NF-nB from not shown). At 20 h post-infection, the expression levels of entering the nucleus and by inhibiting the activity of the kinase both early genes and late genes increased dramatically. complex IKK (Friedman and Horwitz, 2002). Since the Coordinately, the number of differentially expressed host cell transcription factor NF-nB promotes the expression of several genes, as well as the magnitude of regulation, was significantly hundred target genes, mainly involved in the host immune increased. A similar increase was also seen when comparing response (Ghosh et al., 1998), it is anticipated that the differentially expressed host cell genes between 6 and 10 inhibition of the NF-nB pathway leads to deregulated h post-infection. In our previous study, we identified 76 genes expression of many target genes during the adenovirus showing more than 1.5-fold changes using three different infection. cDNA arrays containing totally 12,309 unique genes. Sixteen After the onset of adenovirus DNA replication, viral of these genes were identified on the 7500 cDNA array used in transcription switches from the early regions to late regions. the present investigation. Notably, using the same 1.5-fold Although progression of adenovirus infection results in threshold, 60 and 382 genes were found to be differentially inhibition of cellular DNA replication and protein synthesis regulated at 10 and 20 h post-infection, respectively. Therefore, (Hodge and Scharff, 1969; Huang and Schneider, 1991; Pina we concluded that the regulation of host cell gene expression and Green, 1969), minor effects have been observed on the was augmented as the virus infection progressed into the late transcription of cellular genes (Beltz and Flint, 1979). phase. However, the export of newly synthesized cellular mRNA The genes that were differentially regulated throughout the from the nucleus to the cytoplasm is dramatically reduced early and late phases of adenovirus infection are shown in during the viral late phase (Beltz and Flint, 1979). In contrast, Table 1. For most of these genes, the expression patterns were viral RNAs are efficiently transported to the cytoplasm (Pilder maintained during the early to late transition, although a et al., 1986). The E1B-55 kDa/E4-34 kDa protein complex has slightly increased level of regulation during the late phase was been shown to be required both for blocking cellular mRNA observed. However, three transcription factors, JUNB, NR4A1 transport and facilitating export of viral mRNAs late after and ZNF503, that were upregulated at 6 h post-infection were infection (Bridge and Ketner, 1990; Corbin-Lickfett and downregulated during the late phase of infection. In contrast, Bridge, 2003; Sarnow et al., 1984). In addition, viral mRNAs expression of SSB, encoding the autoantigen La, showed a 238 F. Granberg et al. / Virology 343 (2005) 236–245

Table 1 Genes differentially expressed during the progression of an adenovirus infection Clone ID Symbol 6 ha 10 h 20 h Gene name 50615 HSPA1L 2.0b 4.7 3.5 Heat shock 70 kDa protein 1-like 486678 P2RX5 1.5 1.2 1.8 Purinergic receptor P2X 49970 SSB À2.3 1.2 1.7 Sjogren syndrome antigen B (autoantigen La) 810457 RNPC1 1.8 1.5 1.5 RNA-binding region (RNP1, RRM) containing 1 204301 CDC25A 1.7 1.8 1.3 Cell division cycle 25A 310406 IL6 À1.7 À2.0 À1.4 Interleukin 6 (interferon, beta 2) 1928791 F3 À2.4 À1.2 À1.5 Coagulation factor III 309893 NR4A1 2.1 1.1 À1.5 Nuclear receptor subfamily 4, group A, member 1 810457 ZNF503c 2.3 À1.1 À1.5 Zinc finger protein 503 770670 TNFAIP3 À1.8 À1.9 À1.9 Tumor necrosis factor, alpha-induced protein 3 309864 JUNB 2.1 À1.2 À2.5 Jun B proto-oncogene 365826 GAS1 À1.8 À2.1 À2.7 Growth arrest-specific 1 756405 ID3 À1.5 À2.4 À4.1 Inhibitor of DNA binding 3, dominant negative helix–loop–helix protein 795877 PLK2c À1.5 À2.7 À4.3 Polo-like kinase 2 (Drosophila) 812965 MYC À1.5d À1.9 À7.4 V-myc myelocytomatosis viral oncogene homolog (avian) 232946 SGK À1.7 À3.5 À8.2 Serum/glucocorticoid regulated kinase a Data for 6 h were from previous study (Zhao et al., 2003), using average values obtained from three different arrays. b Fold change in expression between RNA from infected cells compared to RNA from uninfected cells. c PLK2 was named SNK, and ZNF503 was given as MGC2555 in Zhao et al. (2003). d Data obtained from QRT-PCR. downregulation in the early phase but was upregulated during throughout the lytic cycle (Lohr et al., 2003; Zhao et al., 2003). the late phase. Finally, upregulation of CDC25A, a known E1A Expression of additional genes related to growth arrest, such target (Spitkovsky et al., 1996), was sustained up to 10 h post- as GAS1, TOB1 and ELL2, and inhibition of cell cycle infection but diminished at 20 h post-infection. progression ARHE (Villalonga et al., 2004) and were also In addition to an increased number of genes regulated at 10 downregulated. Only two genes, encoding the transcription and 20 h post-infection, we also observed that the average factors AATF and MYBL2, were upregulated. Interestingly, magnitude of regulation increased with the infection time. AATF has a positive effect on growth by relieving the Thus, the 112 genes showing at least a 2-fold change in inhibitory effect of Rb on E2F (Fanciulli et al., 2000) and expression levels in at least one of the two analyzed time points blocking Dlk/ZIP-kinase-induced apoptosis (Page et al., 1999), were identified as specifically regulated during the late phase of whereas MTBL2 plays a stimulatory role in cell cycle infection. It should be noted that approximately 2160 of the progression (Manak et al., 2002). The most dramatically genes printed on the array were detectably expressed in mock- downregulated gene in this group was SGK encoding a infected cells. Using information extracted mainly from serine/threonine protein kinase, which is a potent regulator of SOURCE (database from Genetics Department, Stanford various ion channels and has diverse functions including cell University) and PubMed, known or suggested functions were proliferation and apoptosis (Lang et al., 2003). used to classify 101 identified genes into four categories (listed in Tables 2a–2d). Eleven genes, for which definite functions Sustained downregulation of genes involved in antiviral were unavailable, were excluded. Generally, in contrast to gene defense and stress response regulation during the early phase of infection when a majority of the differentially expressed genes were activated, most genes Similar to the early phase of infection, all of the (82 of the 112) identified in this study were downregulated at differentially regulated genes classified as being involved in 20 h post-infection. the antiviral and stress responses were downregulated (Table 2b). As a central mediator of the immune response, the NF-nB Adenovirus regulation of host cell genes controlling cell cycle pathway has been described as a primary target for many and proliferation is consistent with targeting of growth viruses, including adenovirus. Two of its key players, NFKB2 inhibitory activities (p50) and NFKBIA (InBa), were downregulated. Although NFKBIA is the inhibitor of NFKB, its expression is also Twenty-four genes, classified as regulators of cell cycle and controlled by NF-nB. Within this group, we also identified IL- proliferation, were identified as differentially expressed (Table 6, the IL-6-induced transcription factor CEBPB and the 2a). Several genes encoding transcription factors, including hypoxia susceptibility transcription factor NFE2L2 (Akira et JUNB, ID3, TGFB1I4, KLF4, KLF10, MYC and ETS2, were al., 1990; Chan et al., 2001). Furthermore, LITAF, an important repressed. The transcription factor ETS2 inhibits the cell cycle regulator of tumor necrosis factor alpha (TNFa)gene through activation of the cdk kinase inhibitor p16 (Ohtani et expression, was targeted (Myokai et al., 1999). Correspond- al., 2001). Consistent with earlier microarray experiments, we ingly, the TNFa-induced genes IER3 (Table 2b) and TNFAIP3 found that expression of MYC was persistently downregulated (Table 1) were found to be downregulated. Finally, transcript F. Granberg et al. / Virology 343 (2005) 236–245 239

Table 2a Differentially expressed genes involved in cell cycle and proliferation Clone ID Symbol 10 h 20 h Name 842973 PA2G4 1.3a 2.3 Proliferation-associated 2G4, 38 kDa 815526 MYBL2 1.3 2.2 V-myb myeloblastosis viral oncogene homolog (avian)-like 2 845363 NME1 À1.0 2.2 Non-metastatic cells 1, protein (NM23A) expressed in 2072912 AATF 1.3 2.0 Apoptosis antagonizing transcription factor 266106 YWHAE 1.1 2.0 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide 214133 BRD2 2.1 À1.2 Bromodomain containing 2 188232 KLF4 À1.2 À2.0 Kruppel-like factor 4 725454 CKS2 1.1 À2.1 CDC28 protein kinase regulatory subunit 2 489282 KLF10 À1.5 À2.2 Kruppel-like factor 10 823940 TOB1 À1.3 À2.2 Transducer of ERBB2, 1 811848 TGFB1I1 À1.7 À2.3 Transforming growth factor beta 1 induced transcript 1 309864 JUNB À1.2 À2.5 Jun B proto-oncogene 260303 ETS2 À1.6 À2.5 V-ets erythroblastosis virus E26 oncogene homolog 2 (avian) 365826 GAS1 À2.1 À2.7 Growth arrest-specific 1 784593 ARHE À1.5 À2.8 Ras homolog gene family, member E 898092 CTGF À1.2 À3.1 Connective tissue growth factor 626716 ELL2 À1.7 À3.5 Elongation factor, RNA polymerase II, 2 756405 ID3 À2.4 À4.1 Inhibitor of DNA binding 3, dominant negative helix–loop–helix protein 795877 PLK2 À2.7 À4.3 Polo-like kinase 2 (Drosophila) 504527 DUSP1 À1.4 À4.4 Dual specificity phosphatase 1 868630 TGFB1I4 À2.0 À4.8 Transforming growth factor beta 1 induced transcript 4 812965 MYC À1.9 À7.4 V-myc myelocytomatosis viral oncogene homolog (avian) 840776 SGK À3.5 À8.2 Serum/glucocorticoid regulated kinase a As described in Table 1. levels for several genes induced by diverse stress factors were transport (Table 2c). Several genes controlling cytoskeleton decreased, including MAP2K3, metallothioneins MT1E and organization were downregulated, including ARPC5, a subunit 1H and transcription factor HIF1A. of Arp2/3 complex that nucleates actin filament polymeriza- tion, and TMOD3, which caps the pointed ends of thin actin Deregulation of genes controlling cell architecture and filament. Intermediate filament was also targeted as indicated communication by reduced levels of transcripts of the cytokeratin KRT19. In addition to direct effects on cell structure components, several A notable category of genes targeted during the late phase of genes linking structure to signaling pathways were deregulated infection was related to cell structure, communication and including LMO7, PDLIM7 and VIL2 that act as mediators

Table 2b Differentially expressed genes involved in immune and stress response Clone ID Symbol 10 h 20 h Name 743230 TncRNA À3.4a À7.2 Trophoblast-derived noncoding RNA 897806 HIF1A À1.6 À4.0 Hypoxia-inducible factor 1, alpha subunit (basic helix–loop–helix transcription factor) 246786 CMKOR1 À2.5 À4.0 Chemokine orphan receptor 1 884438 NFE2L2 À1.7 À3.2 Nuclear factor (erythroid-derived 2)-like 2 810724 IER3 À1.5 À3.0 Immediate early response 3 79629 CXCR4 À1.8 À2.9 Chemokine (C-X-C motif) receptor 4 128126 DAF À1.4 À2.9 Decay accelerating factor for complement 208718 ANXA1 À1.4 À2.8 Annexin A1 1472735 MT1E À1.6 À2.7 Metallothionein 1E (functional) 682529 NFKB2 À1.8 À2.5 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) 1461737 CTF1 À1.6 À2.5 Cardiotrophin 1 161993 CEBPB À1.2 À2.4 CCAAT/enhancer binding protein (C/EBP), beta 340734 NFKBIA À1.5 À2.4 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 1358393 MAP2K3 À1.6 À2.3 Mitogen-activated protein kinase kinase 3 214162 MT1H À1.6 À2.1 Metallothionein 1H 745347 LITAF À1.2 À2.0 Lipopolysaccharide-induced TNF factor 310406 IL6 À2.0 À1.4 Interleukin 6 (interferon, beta 2) a As described in Table 1. 240 F. Granberg et al. / Virology 343 (2005) 236–245

Table 2c Differentially expressed genes involved in cellular structure and cell communication Clone ID Symbol 10 h 20 h Gene name 249606 RAB9P40 1.4a 2.2 Rab9 effector p40 2177110 LOC388524 À1.1 2.2 Similar to Laminin receptor 1 197888 GNB2L1 1.0 2.2 Guanine nucleotide binding protein. beta polypeptide 2-like 1 2028912 WASL 1.0 2.1 Wiskott–Aldrich syndrome-like 2305985 GAL 1.3 2.0 Galanin 755145 VIL2 À1.3 À2.0 Villin 2 (ezrin) 85313 CCPG1 À1.4 À2.0 Phosphatidylinositol glycan, class B 52076 OLFM1 À1.3 À2.0 Olfactomedin 1 505203 TMOD3 À1.2 À2.1 Tropomodulin 3 (ubiquitous) 713145 CD44 À1.2 À2.1 CD44 antigen 173674 NPTX1 À1.2 À2.1 Neuronal pentraxin I 810131 KRT19 À1.0 À2.2 Keratin 19 774071 PICALM À1.3 À2.2 Phosphatidylinositol binding clathrin assembly protein 241530 EPHA2 À1.2 À2.2 EPH receptor A2 788256 KIF23 À1.3 À2.3 Kinesin family member 23 110467 CAV2 À1.4 À2.4 Caveolin 2 664975 CLDN1 À1.6 À2.4 Claudin 1 970613 RAD21 À1.2 À2.5 RAD21 homolog (S. pombe) 51582 LMO7 À1.6 À2.6 LIM domain 7 814546 PSCD1 À1.9 À2.9 Pleckstrin homology, Sec7 and coiled-coil domains 1 502682 PDLIM7 À1.5 À2.9 PDZ and LIM domain 7 (enigma) 340558 ARPC5 À1.2 À3.5 Actin-related protein 2/3 complex, subunit 5, 16 kDa 810512 THBS1 À2.6 À6.6 Thrombospondin 1 378488 CYR61 À1.7 À8.4 Cysteine-rich, angiogenic inducer, 61 840567 TM4SF1 À3.5 À18.8 Transmembrane 4 superfamily member 1 a As described in Table 1. between the actin cytoskeleton and the cell adhesion structures UNG and the DNA replication complex factor Pfs2. at the plasma membrane. The tyrosine kinase receptor EPHA2 Components of the RNA metabolic processes included a determines cell malignancies partly by activating the focal kinase (CLK) regulating phosphorylation of serine/arginine- adhesion kinase, and TM4SF1 is a tumor-associated integrin- rich (SR) proteins required for pre-mRNA splicing, as well as binding transmembrane protein. Finally, genes involved in cell- the SR protein SFRS1 (Colwill et al., 1996). Five ribosomal to-cell communications and structure, such as the tight junction proteins, RPL18, RPL35, RPL15, RPL7A and RPS10, were component CLDN1 and the cell surface adhesive glycoproteins increased more than 1.8-fold (data not shown), but only CYR61, CD44 and THBS1, were downregulated. Reduced RPS10 showed more than 2-fold change. Several genes expression of a number of genes encoding structural compo- involved in posttranslational processes, such as protein- nents involved in intracellular transport was also observed: folding, were also identified including two heat shock KIF23 functions as molecular motors for the movements of proteins, HSPA1B and HSPA1L, the hsp90- and hsp70- organelles, microtubules or chromosomes along microtubules; associated protein FKBP and the molecular chaperone CCT7. CCPG1 is a major component of the transverse filaments of It should be noted that 50% of the genes classified as synaptonemal complexes; PICALM is a phosphatidylinositol- involved in metabolism were upregulated, which is striking binding clathrin assembly protein; CAV2 is a major component contrast to the predominant repression of genes within the of the inner surface of the caveolae that mediates endocytosis; categories listed in Tables 2a–2c. and PSCD1 mediates the regulation of protein sorting and membrane trafficking. Taken together, downregulation of these Confirmation of microarray result by quantitative real-time genes might contribute to the disintegration of cell structure PCR (QRT-PCR) and organization observed during the late phase of a virus infection. To confirm the DNA microarray results, QRT-PCR analyses were performed on a subset of genes. For Selective upregulation of genes involved in metabolism and comparison, RNA samples from the 6 h infection were macromolecular synthesis included in the analysis. ACTB was used as normalization factor. The result for five genes, JUNB, MYC, IL-6, MYBL2 Although the majority of deregulated genes demonstrated a and CYR61, confirmed the array data, although the magni- reduced accumulation of RNA during the course of infection, tude of regulation was generally higher in the QRT-PCR expression of several genes linked to macromolecular bioge- compared to the microarray result (Table 3). QRT-PCR was nesis was found enhanced (Table 2d). Among genes involved also performed on the WASL gene, which was the only in the DNA replication were three histones (HIST1H2BJ, upregulated gene involved in actin organization. In contrast to HIST1H2BK and HIST1H2BL), the uracil–DNA glycosylase the microarray results, QRT-PCR analysis of WASL expres- F. Granberg et al. / Virology 343 (2005) 236–245 241

Table 2d Differentially expressed genes involved in cellular metabolism and macromolecular synthesis Clone ID Symbol 10 h 20 h Name 859858 STAR 1.1a 16.4 Steroidogenic acute regulator 290841 HIST1H2BK 1.2 4.1 Histone 1, H2bk 2492634 HSPA1B 4.1 3.6 Heat shock 70 kDa protein 1B 50615 HSPA1L 4.7 3.5 Heat shock 70 kDa protein 1-like 1675553 HIST1H2BJ 1.1 2.6 Histone 1, H2bj 49464 UNG 1.6 2.3 Uracil–DNA glycosylase 2252417 RPS10 À1.1 2.2 Ribosomal protein S10 882484 CCT7 1.2 2.2 Chaperonin containing TCP1, subunit 7 (eta) 884799 CACYBP 1.2 2.2 Calcyclin binding protein 1576468 HSD17B2 1.5 2.2 Hydroxysteroid (17-beta) dehydrogenase 2 281039 FABP5 1.3 2.2 Fatty acid binding protein 5 (psoriasis-associated) 2056049 HIST1H2BL 1.1 2.1 Histone 1, H2bl 1600239 Pfs2 1.3 2.1 DNA replication complex GINS protein PSF2 80399 SFRS1 1.4 2.1 Splicing factor, arginine/serine-rich 1 (splicing factor 2) 1570663 FKBP4 1.5 2.1 FK506 binding protein 4, 59 kDa 712916 PSMC3 1.1 2.1 Proteasome (prosome, macropain) 26S subunit, ATPase, 3 1416782 CKB 1.4 2.1 Creatine kinase, brain 2543376 PGC 1.3 2.1 Progastricsin (pepsinogen C) 813459 GTF2E1 À1.2 À2.0 General transcription factor IIE, polypeptide 1, alpha 56 kDa 205049 HSPB8 À1.2 À2.0 Heat shock 22 kDa protein 8 2449395 AKR1C2 À1.5 À2.0 Aldo-keto reductase family 1, member C2 207358 SLC2A1 À1.3 À2.0 Solute carrier family 2 (facilitated glucose transporter), member 1 485849 CLK1 À1.3 À2.1 CDC-like kinase 1 361639 GCLM À1.0 À2.1 Glutamate-cysteine ligase, modifier subunit 83605 CPS1 À1.4 À2.2 Carbamoyl-phosphate synthetase 1, mitochondrial 2572290 SQSTM1 À1.3 À2.3 Sequestosome 1 705274 DGKD À1.6 À2.3 Diacylglycerol kinase, delta 130 kDa 813187 DDAH1 À1.5 À2.3 Dimethylarginine dimethylaminohydrolase 1 34795 DAZAP2 À1.6 À2.6 DAZ-associated protein 2 48404 GDA À1.2 À2.6 Guanine deaminase 288896 KCNK1 À1.6 À2.7 Potassium channel, subfamily K, member 1 196992 AKR1C1 À1.6 À2.9 Aldo-keto reductase family 1, member C1 1473304 AKR1C3 À1.5 À2.9 Aldo-keto reductase family 1, member C3 252515 KYNU À1.5 À3.4 Kynureninase (l-kynurenine hydrolase) 897733 SLC38A2 À2.0 À5.3 Solute carrier family 38, member 2 789376 TXNRD1 À1.9 À7.7 Thioredoxin reductase 1 a As described in Table 1. sion demonstrated a clear repression, thus concurring with the was increased as the infectious cycle proceeded into the late general repression of genes involved in cell structure phase. Even so, the number of genes still constituted a fraction disintegration. (approximately 5%) of detectably expressed genes, indicating that adenovirus only targets a specific set of cellular genes. Discussion Although it may seem significant that adenovirus targeting of, for example, genes involved in immune response and antivirus In a previous study, we demonstrated that adenovirus had a defense was solely manifested by downregulation, whereas half limited effect on the expression of host cell genes during the of the targeted genes related to macromolecular biogenesis was early phase of infection in HeLa cells. Here, we show that the upregulated, expression of the majority of host cell genes was number of target genes, as well as the magnitude of regulation, left untouched.

Table 3 Confirmation of microarray results by QRT-PCR Symbol Name QRT-PCR Microarray 6 h p.i. 10 h p.i. 20 h p.i. 10 h p.i. 20 h p.i. JunB junB proto-oncogene 1.5a À1.3 À21.40 À1.2 À2.5 CYR61 Cysteine-rich, angiogenic inducer, 61 1.2 À1.5 À46.6 À1.7 À8.4 MYC V-myc myelocytomatosis viral oncogene homolog (avian) À1.5 À2.6 À23.6 À1.9 À7.4 IL-6 Interleukin 6 À3.4 À3.2 À32.7 À2.0 À1.4 MYBL2 V-myb myeloblastosis viral oncogene homolog (avian)-like 2 À1.1 1.3 2.9 1.3 2.2 WASL Wiskott–Aldrich syndrome-like À1.3 1.2 À3.0 1.0 2.1 a As described in Table 1. 242 F. Granberg et al. / Virology 343 (2005) 236–245

During the later stages of infection, adenovirus employs releasing E2F (Zhao et al., 2003). After efficient replication of additional strategies to redirect the cellular synthesis machinery the viral genome has been obtained, regulation of E2F- to virus production only; the E1B-55 kDa/E4-34 kDa complex dependent genes may no longer be of prime importance. This mediates a preferential export of viral mRNA, and the L4-100 might be exemplified by the upregulated expression of the cell kDa protein selectively inhibits translation of cellular mRNA. cycle regulating phosphatase CDC25A specifically during the The use of several strategies to ensure optimal synthesis of early phase. During the late phase, it is likely that more efforts viral products is likely to be relevant during the non-optimized are made to inhibit cellular attempts to halt growth and induce conditions of infection in natural host organisms. Whereas the apoptosis. E1A has previously been shown to block growth majority of cellular target genes were upregulated at 6 h post- inhibition enforced by TGF-h signaling pathway (de Groot et infection, the regulation during the late phase primarily caused al., 1995), and, at 6 h post-infection, we observed an a decline in gene expression. A limited number of genes were, upregulation of SMURF1, a TGF-h signal terminator, which however, upregulated at 20 h post-infection. Most of these triggers the degradation of SMAD1 and 5 (Zhao et al., 2003). genes appeared refractory to export inhibition of cellular During the late phase of infection, expression of several TGF-h mRNA since upregulation was also observed in the cytoplas- inducible genes was reduced, including JUNB, ID3, MYC, mic fraction (Zhao et al., unpublished observation). This KLF10, TGFB1I4, TGFB1I1, IL-6, LMO7, SGK and CTGF. supports the hypothesis that mRNA export is authorized by Of these genes, JUNB, ID3, KLF10, KLF4 and TGFB1I4 have transcriptional activation during the late phase (Yang et al., been described as transcriptional repressors that block cellular 1996). It remains to be determined whether these mRNAs proliferation (Chen et al., 2002). TGFB1I1 and CTGF are both continue to be translated even when accumulation of L4-100 K lost during tumor progression (Martinerie et al., 1992; has displaced the eIF4E kinase from the cap-initiation complex Shibanuma et al., 1994). Consistent with the inhibition of the eIF4F (Cuesta et al., 2000). TGF-h signaling pathway, we also found that expression of A gradual switch from upregulation to downregulation was AATF, a target for TGF-h repression (Lindfors et al., 2000), observed during the 6 to 20 h post-infection interval. Several was upregulated during the late phase. Through interacting mechanisms where viral DNA replication provides the break with pRb, AATF may, together with E1A, play an important point might account for this altered pattern of regulation. (i) role in releasing the pRb inhibition of E2F (Cobrinik, 1996; During the transit from early to late phase, the viral DNA Fanciulli et al., 2000). Targeting of the TGF-h is unlikely to be replication results in a dramatic increase in transcription- the only way to avoid growth repression since several TGF-h- competent templates that are able to compete for the cellular independent growth-arrest-inducing genes, such as GAS1, transcription machinery and titrate cellular repressors (Iftode TOB1, ARHE and DUSP1, were also repressed (Alessi et al., and Flint, 2004). (ii) The activation of a late specific viral 1993; Del Sal et al., 1992; Matsuda et al., 1996; Villalonga et transcription pattern may also affect the utilization of cellular al., 2004). promoters. (iii) Expression of proteins from the major late Expression of SSB was downregulated during the early time transcription unit has been implicated in regulation of viral gene (Table 1) but gradually increased following the progression expression and turning off expression of cellular genes. (iv) into the late phase of infection. SSB encodes the La Inhibition of cellular mRNA transport may result in decreased autoantigen that has been shown to stabilize histone mRNA accumulation due to altered RNA stability. (v) The immediate (McLaren et al., 1997). It remains to be determined whether the early gene E1A preferentially encodes the 289R major increased levels of mRNA for three histone 2B genes transcriptional activator during the early phase. As the infection (HIST1H2BJ, HIST1H2BK and HIST1H2BL) is caused by proceeds into the late phase, a shift in splice selection results in an SSB-dependent increase in half-life. In addition, it is also an increased accumulation of the shorter E1A-243R protein possible that the upregulation of SSB is required to maximize (Chow et al., 1979). Although E1A-243R retains its capacity to the accumulation of adenoviral mRNA. The La protein also has bind pRb, thereby releasing E2F (Dyson et al., 1992), this a positive effect on the translation of mRNAs characterized by smaller variant of E1A mainly functions as a transcriptional a5Voligopyrimidine sequence (5V-TOP) (Crosio et al., 2000). repressor through its capacity to bind the transcriptional Genes encoding ribosomal proteins are TOP genes, and we integrator p300/CBP (Jones, 1995). Since p300/CBP functions found that transcripts of six ribosomal proteins, RPL18, as a coactivator for numerous transcription factors, it is possible RPL35, RPL15, RPL7A and RPS10, were all upregulated. that the ability of E1A to sequester p300/CBP contributes to the Therefore, it is possible that enhancing expression of genes downregulation of cellular genes observed during the late phase encoding ribosomal proteins occurs at both transcription and of infection. (vi) Finally, it is also possible that the upregulation translation levels. of cellular genes during the early phase, such as the transcription The last stages of an adenovirus infection are distinguished factors JUNB, NR4A1 and ZNF503, may affect downstream by strong morphological reorganization of host cell structure targets at later stages of infection. generally described as the cytopathic effect. Starting already Regulating expression of genes required for S-phase entry is during the early phase of infection, the integrity of one of the major events during the early phase of adenovirus intermediate filaments is disrupted, and vimentin-like filament infection. The over-representation of putative E2F binding sites collapses into the perinuclear region. Late in the infection, in adenovirus-induced host cell genes at 6 h post-infection is keratins, such as keratin18, are also cleaved by viral protease consistent with the ability of E1A to interact with pRb, thereby (Chen et al., 1993). In addition to proteolysis, our data F. Granberg et al. / Virology 343 (2005) 236–245 243 demonstrated a decreased accumulation of RNA from genes adenovirus type 2 at a multiplicity of 100 fluorescence-forming involved in cell architecture. Downregulation was seen in units per cell (Philipson, 1961) in a serum-free DMEM. After genes involved in actin and intermediate filaments organiza- 45 min of adsorption at 37 -C, the medium was replaced with tion, communication between actin and the plasma membrane, DMEM containing 10% NCS. At 10 and 20 h after infection, intercellular contacts and cell adhesion triggered signals. total cellular RNA was extracted using TRIZOL Reagent Destruction of the cell integrity most likely makes the (Invitrogene). Total RNA was prepared from two independent infected cell more susceptible to lysis and apt to release adenovirus and mock infections of HeLa cells, respectively. progeny virus. In addition, disruption of cell–cell adhesion The quantity and quality of RNA were determined using the and tight junctions might allow the virus to spread directly RNA 6000 Nano LabChip Kit and a Bioanalyzer 400 (Agilent between cells deeper into the infected tissue, thereby evading Technologies). circulating inflammatory cells. In a previous report, we have analyzed regulation of gene Preparation of cDNA probe and microarray hybridization expression in HeLa cells during infection with the oncogenic adenovirus type 12 (ad12) (Dorn et al., 2005). A lytic ad12 Microarrays containing 7500 duplicate-printed human infection takes twice the time, is less productive and causes cDNAs (Human Sequence Verified Set from Research Genetics reduced cytopathic effect compared to an ad2 infection (23 Huntsville, AL, USA) were obtained from the DNA Micro- and reference therein). Overall, more genes were found to be array Core Facility, Uppsala University, Sweden. In order to regulated during the ad2 infection (79 cellular genes showed monitor the progression of infection, adenovirus early genes more than 1.5-fold regulation during the course of ad12 E1A, E1B, E2A, E3 and E4 and late gene L1 and L3 were also infection compared to 382 genes showing more than 1.5-fold printed on this array (Zhao et al., 2003). Ten micrograms of change from 10 to 20 h post-infection with ad2). Still, over total RNA from adenovirus- and mock-infected cells was used 55% of the genes identified from the analysis of the ad12 for generating biotin-labeled cDNA, which was then coupled infection were shown to be differentially expressed also in with either Cy5 or Cy3, respectively, and hybridized to the ad2-infected cells. Genes involved in immune response, cell cDNA microarray according to the manufacturer’s post- cycle/proliferation and metabolism were main targets for both labeling protocol (GE Health Care). To control for dye biases, viruses. However, a few clear differences were found. Most duplicate reactions were carried out where the fluorescent dyes TGF-h-inducible genes, as well as genes involved in the were switched during the coupling reaction (so called ‘‘dye- TGF-h signaling pathway, were only identified in ad2- swap’’ labeled). Thus, a total of 8 experiments were performed, infected cells. Although it is possible that some of the 4 arrays for 10 h and 4 arrays for 20 h RNA samples. differences might be the result of the lower number of genes found to be regulated during an ad12 infection, it is tempting Data analysis to speculate that the observed differences constitute the basis for the different growth characteristics of ad12 and ad2. Data collection and normalization were done as described Finally, the splicing factor SFRS1 was upregulated in ad2- before (Zhao et al., 2003). Briefly, spot intensity was quantified infected cells but downregulated in ad12-infected cells. using the GenePix Pro 5.1 software. The data, including SFRS1 is required for adenovirus RNA splicing by enhancing corresponding images, were stored in the BASE database at the the selection of a proximal 5Vsplice site in E1A mRNA during Linnaeus Centre for Bioinformatics (LCB), Uppsala University the early phase. However, during the late phase, the SFRS1 (Saal et al., 2002). Data handling and print-tip intensity- protein is dephosphorylated which allows altered splice site dependent normalization (Yang et al., 2002) was performed for selection (Gattoni et al., 1991; Himmelspach et al., 1995). each experiment within the framework of LCBs Dataware- Although upregulation of SFRS1 seems contra-productive house. Differentially expressed genes in adenovirus-infected from the splicing perspective, recent data indicate that cells, as compared to mock-infected cells, were identified by unphosphorylated SFRS1 promotes export of spliced mRNA employing cut-off criteria of average-fold value larger than 2 (Huang et al., 2004). Notably, the late infected cell nucleus is and a coefficient of variation (CV) less than 50% in any of the loaded with viral mRNA to be exported. Alternative splicing two time points. in ad12 is essentially unexplored, and it also remains to be investigated whether the ad12 have developed specific export Quantitative real-time PCR (QRT-PCR) mechanisms. To verify the results obtained by the microarray experiment, Materials and methods the QRT-PCR analyses were performed on the same sets of RNA samples used for microarray hybridization. PCR primers Cell culture, virus infection and RNA isolation and Taqman MGB probes for the following genes were purchased from Applied Biosystems: MYC, JUNB, ACTB, HeLa cells were grown in Dulbecco’s modified Eagle’s MYBL2, WASL and CYR62. For cDNA synthesis, 2 Agof medium (DMEM) supplemented with 10% newborn calf serum RNA was reverse-transcribed using Omscript (Qiagen). Real- (NCS), 100 U/ml penicillin and 100 Ag/ml streptomycin. time PCR was performed as described previously (Zhao et al., Subconfluent cells were mock-infected or infected with 2003). 244 F. Granberg et al. / Virology 343 (2005) 236–245

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