Review

Hypoxia-inducible factor 1 and its role in viral carcinogenesis

Sean Cuninghame a,b, Robert Jackson a,b, Ingeborg Zehbe a,b,n

a Probe Development & Biomarker Exploration, Thunder Bay Regional Research Institute, 980 Oliver Rd, Thunder Bay, Ont., Canada P7B 6V4b Department of Biology, Lakehead University, Thunder Bay, Ont., Canada

a r t i c l e i n f o

Article history:Received 10 December 2013Returned to author for revisions17 January 2014Accepted 26 February 2014Available online 1 April 2014

Keywords:HIF-1OncovirusesMetabolismAngiogenesisInvasionMetastasisTumor microenvironment

a b s t r a c t

The advent of modern molecular biology has allowed for the discovery of several mechanisms by whichoncoviruses promote carcinogenesis. Remarkably, nearly all human oncogenic viruses increase levels of thetranscription factor hypoxia-inducible factor 1 (HIF-1). In this review, we highlight HIF-1's significance inviral oncogenesis, while providing an in-depth analysis of its activation mechanisms by the followingoncoviruses: human papillomaviruses (HPVs), hepatitis B/C viruses (HBV/HCVs), Epstein–Barr virus (EBV),Kaposi's sarcoma-associated herpes virus (KSHV), and human T-cell lymphotropic virus (HTLV-1). Wediscuss virus-induced HIF-1's role in transcriptional upregulation of metabolic, angiogenic, and microenvir-onmental factors that are integral for oncogenesis. Admittedly, conclusive evidence is lacking as to whetheractivation of HIF-1 target genes is necessary for malignant transformation or merely a result thereof. Inaddition, a complete understanding of host–virus interactions, the effect of viral genomic variation, and theclinical (and potential therapeutic) relevance of HIF-1 in viral oncogenesis warrant further investigation.

& 2014 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371Oncogenic viruses enhance HIF-1α levels by modulating its transcription, translation, or stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

The human papillomaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372The hepatitis viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374The oncogenic herpes viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375The human T-cell lymphotropic virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

Downstream activation of genes involved in virally-induced cancer progression by HIF-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378Invasion and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

Cell adhesion and motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379The microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/yviro

Virology

http://dx.doi.org/10.1016/j.virol.2014.02.0270042-6822 & 2014 Elsevier Inc. All rights reserved.

Abbreviations: 4E-BP, 4E binding protein; AP-1, activator protein-1; Ad-AH, EBV-negative adenocarcinoma cell line; Akt, serine/threonine kinase; ARNT, aryl hydrocarbonreceptor nuclear translocator; ATP/ADP, adenosine tri- or diphosphate; CAIX and CAXII, carbonic anhydrase IX and XII; E-cadherin, epithelial adhesion molecule; E6-AP, E6-associated Protein (E3 ubiquitin ligase); EBNA, EBV nuclear antigens; EBV, Epstein–Barr virus or HHV-4; eIF-4E, elongation initiation factor 4E; EMT, epithelial–mesenchymaltransition; ERBB2, growth factor receptor; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT-1, GLUT-3, glucosetransporters; HBV/HCV, hepatitis B or C virus; HCC, hepatocellular carcinoma; HDAC, histone deacetylases; HHV, human herpes virus; HIF, hypoxia-inducible factor; HK1/2,hexokinase 1 & 2; HPV, human papillomavirus; HRE, hypoxia response element; Huh-7, cell type permissive to infection by HCV; IKKβ, inhibitor of nuclear factor kappa-Bkinase subunit beta; KSHV, Kaposi's sarcoma-associated herpes virus or HHV-8; LANA, latent nuclear antigen, LANA-2 is the viral homolog of cellular interferon regulatoryfactor 3; LCL, lymphoblastoid cell lines, created by EBV-transformed B cells; LDHA, lactate dehydrogenase; LMP, EBV latent membrane proteins; MAPK, mitogen-activatedprotein kinase; MEK, activator of ERK (kinase); MMP, matrix metalloprotease; MNK, MAPK-interacting kinase; mTOR, mammalian target of rapamycin, a PI3K; NF-κB,nuclear factor kappa-light-chain-enhancer of activated B cells; ODD, oxygen-dependent domain of HIF-1; ORF, open reading frame; PA-1, cell type permissive to infection byHPV; PDK1, pyruvate dehydrogenase kinase 1; PFK, phosphofructokinase; PGK1, phosphoglycerate kinase 1; PHD, prolyl hydroxylase; PI3K, phosphatidylinositol-3-kinase;PTEN, tumor suppressor; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SUMO, small ubiquitin-like modifier; SP-1, specificity protein-1; STAT-3, signaltransducer and activator of transcription 3; TCA, mitochondrial tricarboxylic acid; URR, upstream regulatory region; VEGF, vascular endothelial growth factor; vGPCR, viral G-protein coupled receptor; VHL, von Hippel Lindau E3 ligase protein

n Corresponding author at: Probe Development & Biomarker Exploration, Thunder Bay Regional Research Institute, 980 Oliver Road, Thunder Bay, Ont., Canada P7B 6V4.Tel.: þ1 807 684 7246.

E-mail address: zehbei@tbh.net (I. Zehbe).

Virology 456-457 (2014) 370–383

Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

Introduction

Approximately 16% of yearly cancer diagnoses worldwide areattributable to an infectious agent (de Martel et al., 2012). The fractionof cancers linked to infection varies greatly by geographical locationand socioeconomic factors: while only 7.4% of cancer diagnoses are theresult of infection in developed countries, up to 22.9% of cancers indeveloping parts of the world arise due to infection. In Sub-SaharanAfrica, a striking one-third of cancer diagnoses are the result ofinfection (de Martel et al., 2012); the majority of these cancers arethe result of infection with an oncovirus. Thus, oncovirus-inducedcancers are an important global concern.

Oncoviruses contribute to carcinogenesis by altering the func-tion of cellular targets that play pivotal roles in the development ofcancer. Over the past few decades, many of the cellular mechan-isms by which oncoviruses induce malignant transformation havebeen elucidated. One of the best-documented cases of oncogenicviruses disrupting normal cellular functions involves the tumorsuppressor protein, p53. Several oncogenic viral proteins—such asthe E6 oncoprotein of high-risk human papillomavirus (HPV) typesand the HBx oncoprotein of hepatitis B virus (HBV)—have inhibi-tory effects on the pro-apoptotic ability of p53. The high-risk HPVshave additional cellular targets: the E6 oncoprotein increasestelomerase activity and the E7 oncoprotein degrades the retino-blastoma tumor suppressor protein (Scheffner et al., 1993; Boyeret al., 1996; Klingelhutz et al., 1996).

There is strong evidence that activation of the hypoxia-induciblefactor 1 (HIF-1) transcription factor is a common pathway affected

by human oncogenic viruses. HIF proteins are major components ofthe innate hypoxic stress response in non-cancerous cells, acting astranscription factors for a multitude of genes required for adaptationunder low oxygen conditions (Wang et al., 1995a). Three HIF isoformshave been identified (i.e., HIF-1, HIF-2 and HIF-3), however researchto date has focused primarily on HIF-1. As such, this review will focuson HIF-1, but HIF-2 and HIF-3 could be assessed in future studies ofviral carcinogenesis.

Transcriptionally active HIF-1 is a heterodimer made up ofα- and β-subunits (the β subunit is also known as aryl hydro-carbon receptor nuclear translocator (ARNT), which also dimerizeswith several other transcription factor subunits). The dimer is amember of the basic helix loop helix-PER-ARNT-SIM (bHLH-PAS)family of transcription factors that play a role in cancer develop-ment (Bersten et al., 2013). In normal, non-hypoxic cells, HIF-1α iscontinually synthesized and degraded, while HIF-1β is constitutivelyexpressed to levels that remain relatively constant within thenucleus (Fig. 1A). HIF-1 activity is largely dependent on theregulation of its α subunit at several levels including transcription,translation, ubiquitin-mediated protein breakdown, nuclear trans-location, and association with transcriptional co-activators. HIF-1αmRNA is ubiquitously expressed and its levels are similar for mostcell types studied between hypoxic and normoxic conditions(Wenger et al., 1997). However, some cell types, such as hepato-cellular carcinoma (HCC) Hep3B cells, exhibit an increase in HIF-1αtranscription under hypoxic conditions (Wang et al., 1995a). In vivoinvestigations initially uncovered the potential for environmentalhypoxia inducing HIF-1α transcription (Wiener et al., 1996), but

Fig. 1. Regulation of HIF-1 protein levels. The level of HIF-1 in a given cell is subject to its oxygen-independent synthesis (A) and oxygen-dependent degradation (B). (A) Thesynthesis of HIF-1α is augmented in an oxygen-independent manner by transcription factors acting on its upstream regulatory region (URR) and the PI3K/Akt and ERK/MAPKpathways acting on its translation. When a growth factor binds to its respective tyrosine kinase receptor, PI3K (in the PI3K pathway) or RAS (in the ERK/MAPK pathway) isactivated. In the PI3K pathway, PI3K then activates Akt and subsequently mTOR. In the ERK/MAPK pathway, RAS activates RAF, which activates MEK and subsequently ERK.ERK activates MNK that, together with mTOR, deactivates the 4E-BP, which allows for the formation of the eIF-4E complex. Along with p70S6K1, that is also activated by ERK,eIF-4E enhances the translation of HIF-1α mRNA. (B) When oxygen is present, PHDs are active and hydroxylate HIF-1α at Pro-402 and/or Pro-564. HIF-1α-OH becomesubiquitinylated by VHL and subsequently broken down by the 26S proteasome. Conversely, under hypoxic conditions, PHDs cannot hydroxylate HIF-1α and it will accumulatein the cytoplasm before translocating to the nucleus to complex with HIF-1β and p300/CBP to enhance target gene expression. A rectangular border represents nucleic acidsand circles are proteins. Black and white backgrounds represent specific HIF-1 and effector cellular components, respectively. The dotted line represents the nuclearmembrane. Refer to abbreviation list for full names.

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conflicting studies found no such effect (Wenger et al., 1996;Stroka et al., 2001). More recent work has supported the notionthat hypoxia can influence HIF-1α transcription through inter-mediate pathways (Sperandio et al., 2009; BelAiba et al., 2007).Alternatively, analysis of the promoter and 50-UTR of the HIF-1αgene revealed putative binding sites of several transcriptionfactors in this region including specificity protein (SP-1), activatorprotein (AP-1), nuclear factor kappa-light-chain-enhancer of acti-vated B cells (NF-κB) and HIF-1 itself (Minet et al., 1999). Recentstudies found that enhancing NF-κB and signal transducer andactivator of transcription 3 (STAT-3) increase the transcription ofHIF-1α (van Uden et al., 2008; Rius et al., 2008; Niu et al., 2008).

While HIF-1α transcription is continual, several growth factorsand their associated pathways play a role in enhancing HIF-1αsignaling in an oxygen-independent manner by augmenting thetranslation of HIF-1α mRNA. In the phosphatidylinositol-3-kinase(PI3K) pathway, binding of a growth factor (e.g., insulin-likegrowth factor 1) to its cognate tyrosine kinase receptor (RTK)results in phosphorylation of PI3K, which in turn, activates theserine/threonine kinase, Akt (an action that can be inhibited by thetumor suppressor, PTEN), and subsequently phosphorylates mam-malian target of rapamycin (mTOR) (Semenza, 2003). Additionally,growth factors can act on the mitogen-activated protein kinase(MAPK) pathway such that MAPK kinase (MEK) is activated by RAFand goes on to phosphorylate MAPK (ERK). Activated ERK is thencapable of phosphorylating p70S6K1, which is required for trans-lation of HIF-1α mRNA. In addition to activation of p70S6K1, ERKalso activates MAPK-interacting protein (MNK) that functions toactivate the translation initiator, elF-4E together with mTOR byinhibiting the 4E binding protein (4E-BP) (Agani and Jiang, 2013).In this fashion, the PI3K and MAPK pathways collectively functionto initiate HIF-1α protein synthesis in an oxygen-independentmanner (Fig. 1A).

The majority of work surrounding HIF-1 regulation has been onits constitutive normoxic protein breakdown. HIF-1α degradationis initiated by hydroxylation at one of its proline residues (Pro-402and/or Pro-564) by prolyl hydroxylases (PHD-1, PHD-2, and PHD-3) that use molecular oxygen as a co-substrate (Bruick andMcKnight, 2001; Semenza, 2001). Upon hydroxylation, HIF-1α-OH becomes ubiquitinylated by the von Hippel Lindau E3 ubiquitinligase protein (VHL), and subsequent proteasomal breakdownoccurs. When there is a lack of oxygen, PHDs cannot function,resulting in stabilization of HIF-1α in the cytoplasm. Once HIF-1αaccumulates in the cytoplasm it may translocate to the nucleus byway of α/β importins that requires a nuclear localization signal(NLS) sequence in HIF-1α's C-terminus transactivation domain(Depping et al., 2008). Once in the nucleus, HIF-1α may dimerizewith HIF-1β followed by HIF-1 complexing with the transcrip-tional co-activators p300/CBP. Several studies have found thatphosphorylation of HIF-1α by way of the MAPK pathways enhancestability and may also be required for transactivation (Richardet al., 1999; Suzuki et al., 2001; Liu et al., 2003) in addition to otherposttranslational modifications that alter HIF-1 function includingacetylation (Lim et al., 2010) and small ubiquitin-like modifier(SUMO)ylation (Cheng et al., 2007). Furthermore, complex forma-tion is inhibited under hypoxic conditions by the hydroxylase,factor inhibiting HIF-1 (FIH-1). Similar to the PHDs, FIH-1 hydro-xylates the transactivation domain of HIF-1α at Asn-803, whichinhibits the binding of the heterodimer HIF-1 to its transcriptionalcoactivator p300 (Lando et al., 2002). If each of these HIF-1-regulatory checkpoints are overcome in a given cell, activated HIF-1 binds to hypoxia response elements (HREs) in target genepromoters, transcriptionally activating a multitude of genes withspecific roles in the hypoxic response (Fig. 1B). Soon after theseminal discovery of HIF-1 by Semenza and colleagues in the early1990s, it was found that its overexpression was common in a

variety of cancers (Zhong et al., 1999; Krieg et al., 2000). Moreover,increased HIF-1 has been associated with an unfavorable prog-nosis in most cancers, as it activates genes that play a role inpromoting cancer metabolism, angiogenesis, invasion, and metas-tasis (Semenza, 2003, 2010, 2012). Originally, this increase of HIF-1α in cancers was primarily attributed to stabilization by environ-mental hypoxia or genetic mutations (e.g., to VHL) in the pathwaysleading to the destruction of HIF-1α (Semenza, 2003). However,it has now become clear that most, if not all, human oncogenicviruses directly enhance HIF-1, through various mechanismsaffecting its activity. Thus, HIF-1 represents a common pathwayaffected by these genetically unrelated viruses.

In this review, we will outline the current understanding of themechanisms by which oncogenic viruses promote HIF-1 activity,provide evidence for HIF-1's activation by oncoviruses in theupregulation of key downstream target genes involved in cancerprogression, and highlight the activation of this pathway as aunifying event in viral-induced malignant transformation.

Oncogenic viruses enhance HIF-1α levels by modulating itstranscription, translation, or stabilization

The inhibition of HIF-1α's transcriptional activity in non-cancerous, normoxic cells, involves several processes—obstructionof any one of the sequential steps in HIF-1α destruction can leadto incomplete HIF-1α breakdown and subsequent activation ofdownstream genes. Furthermore, it appears as though an increasein HIF-1α mRNA translation via the PI3K/Akt/mTOR or MAPKsignaling pathways can also lead to an increase in activation oftarget genes, such that the rate of proteasomal breakdown by theoxygen-dependent pathway of HIF-1α is insufficient to inhibit itstranscriptional activity (Jiang et al., 2001; Laughner et al., 2001).It is obvious that there exist multiple ways by which HIF-1 activitycan be increased; each one of these pathways can potentially betargeted by oncogenic viruses for production of gene productsconducive to cancer cell growth and spread (Tables 1 and 2 andFig. 2). Whatever path may be taken, it is clear that six of the sevenknown oncogenic viruses have a component in their infectiousarsenal that results in HIF-1 activity.

The human papillomaviruses

Human papillomaviruses (HPVs) were first described in themid-1980s as a causative agent of cervical cancer (zur Hausen,2002), which could be either malignant (high-risk types) or benign(low-risk types). Several putative cellular targets of the two HPVoncogenes (E6 and E7) have been characterized, including p53,pRb (reviewed in Wise-Draper and Wells, 2007), and morerecently, HIF-1. The first evidence of HPV inducing HIF-1 activitycame from in vivo studies of K14-HPV16 transgenic mice whichharbor the entire early region of HPV16 under the control of thebasal keratin 14 promoter. The authors reported an upregulation ofHIF-1α and its target mRNAs during epidermal carcinogenesis(Elson et al., 2000). The use of an in vivo model provided a contextreminiscent of HPV infection where early genes are expressed inbasal keratinocytes. However, in this particular study, quantifica-tion of viral gene expression and protein levels were not assessedfor their correlation with the HIF-1 pathway. The authors con-tinued these in vivo studies further characterizing the role of HIF-1in cervical carcinogenesis (Lu et al., 2007). Along with these in vivoassessments of the role of HIF-1 in HPV16 carcinogenesis, both theE6 and E7 oncogenes of high risk HPVs have been scrutinized fortheir ability to induce HIF-1 transcriptional activity.

The E6 oncogene has many well-studied cellular functions,including the proteasomal breakdown of p53 via the E3 ubiquitin

S. Cuninghame et al. / Virology 456-457 (2014) 370–383372

Table 1Mechanisms of oncoviral induced HIF-1α enhancement (by virus).

Oncovirus(type)

Associatedcancer(s)

Viral protein(s) Cellular target(s) Mechanism/context Reference

HPV(dsDNA)

Ano-genital,HNSCC, Others

Early region N/A HPV16 transgenic mice have higher levels of HIF-1α and target mRNAs Elson et al. (2000)

E6 p53 E6-mediated p53 degradation enhances HIF-1α stability Ravi et al. (2000)E6, E7, wholegenome

N/A Hypoxia mimetic DFO treatment enhances HIF-1α stability in cells transfected with whole genome or transducedwith E6 and E7 either separately or together

Nakamura et al.(2009)

E7 HDAC1, 4, 7 E7 binds to HDACs that associate with HIF-1α, enhancing transcription complex formation Bodily et al. (2011)

HBV(dsDNA-RT)

HCC HBx ERK1/2 MAPK pathway The MAPK pathway enhances the protein synthesis of HIF-1α independent of oxygen levels Yoo et al. (2003)

HIF-1α Binding by the C-terminus of HBx interferes with PHD/VHL-mediated HIF-1α protein degradation Yoo et al. (2004)Moon et al. (2004)

HDAC1 Enhancing HDAC1 transcription leads to increased deacetylation of the ODD of HIF-1α, inhibiting its degradation Yoo et al. (2008)

HCV(ssRNA)

HCC Subegenomicrepliconregion of HCVgenotype 2a

PI3K/Akt, ERK1/2 MAPK,STAT-3, NF-ĸB pathways

HCV infected Huh-7 cells indirectly activate several kinase pathways acting on HIF-1α by increasing intracellularoxidative stress

Nasimuzzamanet al. (2007)

Core NF-ĸB HCV Core protein enhances NF-ĸB signaling leading to HIF-1α mRNA transcription Abe et al. (2012)

EBV(dsDNA)

Lymphomas, NPC LMP-1 ERK1/2 MAPK pathway LMP-1 enhances the rate of protein synthesis of HIF-1α by enhancing ERK1/2 MAPK signaling Wakisaka et al.(2004)

PHD-1, LMP-1 upregulates Siah-1, an E3 ubiquitin ligase that targets PHD-1 and PHD-3 for breakdown Kondo et al.(2006)PHD-3

EBNA-1 Activation of the AP-1transcription factor

EBNA-1 enhances the transcription of the HIF-1α gene, potentially by increasing the activity of AP-1, O’Neil et al.(2008)

EBNA-5, PHD-1, EBNA-3 and EBNA-5 bind to PHD-1 and PHD-2, respectively, inhibiting the breakdown of HIF-1α via the oxygen-dependent pathway

Darekar et al.(2012)EBNA-3 PHD-2

KSHV(dsDNA)

Kaposi’s Sarcoma vGPCR ERK1/2 and p38 MAPKpathways

ERK1/2 and p38 MAPK both act as independent pathways in the phosphorylation (activation) of HIF-1α Sodhi et al. (2000)

PI3K/Akt, ERK1/2, p38,IKKβ

vGPCR expressing endothelial cells secrete cytokines acting on several pathways enhancing the synthesis of HIF-1α Jham et al. (2011)

LANA-2/vIRF-3 HIF-1α vIRF3 binds directly to HIF-1α, leading to an increase in nuclear translocation and activity Shin et al. (2008)LANA1 VHL LANA1 functions as part of the EC5S ubiquitin complex to degrade VHL Cai et al. (2006a)

HTLV-1(ssRNA-RT)

T-Cell Leukemia Tax PI3K/Akt pathway Under normoxic conditions, HTLV-1 infected cell lines had high levels of HIF-1α Tomita et al.(2007)

Abbreviations: HNSCC, head and neck squamous cell carcinoma; HCC, hepatocellular carcinoma; NPC, nasopharyngeal carcinoma; ODD, oxygen-dependent domain; see text for full names of abbreviated viruses, viral proteins, andcellular targets.

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373

ligase, E6-Associated Protein (E6-AP). The p53 tumor suppressorprotein binds to and induces HIF-1α degradation thus the pre-sence of E6 induces an increase in HIF-1α under hypoxic condi-tions (Ravi et al., 2000). In their study, Ravi and colleaguesconfirmed that transfection of PA-1 ovarian teratocarcinoma cellswith the high-risk HPV E6 oncogene boosts HIF-1α stability underhypoxic (1% O2) culture conditions. While this study shows thatE6-mediated p53 breakdown enhances HIF-1α, it is not clearwhether or not this is reminiscent of an HPV infection as only E6was introduced and levels of it's DNA copy number, mRNA andprotein expression were not assessed. Although E6 was introducedinto atypical host cells, the study set the stage for the investigationof HPV and HIF-1α in normal keratinocytes.

The cell cycle progression-altering E7 oncogene of both high-risk (type 16, 18, 31) and low-risk (type 11) HPV increases HIF-1activity in the presence of several histone deacetylases (HDACs) bybinding to HIF-1α directly (Bodily et al., 2011). The binding of E7 toHIF-1α was directly inhibited through deletion of the N-terminuspRb binding domain of E7. Many HDACs (including HDAC1, 4, and7) enhance HIF-1 activity on their own (Kato et al., 2004; Qianet al., 2006; Yoo et al., 2006), however the authors hypothesizedthat binding of HPV E7 to HIF-1α allows for the displacement ofthese HDAC's from HIF-1α, permitting formation of the HIF-1transcription complex, leading to downstream activation of targetgenes. Previously, under conditions mimicking hypoxia, cellstreated with the hypoxic mimetic, deferoxamine, plus high-riskHPV E7 or E6 exhibited higher protein levels of HIF-1α incomparison to non-transduced keratinocytes under the sameconditions (Nakamura et al., 2009). The authors also looked atwhole genome transfection of high risk (HPV31) and low risk(HPV11) viruses and found that HIF-1α protein levels wereenhanced under hypoxic conditions. However, a full evaluationof viral mRNA and protein expression was not included. Thus, themagnitude of viral gene expression required for an observedincrease in HIF-1α protein under these conditions by HPV is notconclusive. These findings held true for HPV16 E6 and E7 undernormoxic conditions when transfected into HeLa (HPV18þ) andC33A (HPV-) cervical cancer cell lines (Tang et al., 2007). Futurestudies regarding the effect of HPV on the HIF-1 pathway arewarranted in order to establish a quantifiable relationship betweenHPV expression and HIF-1 activity.

The hepatitis viruses

Infection with either hepatitis B (HBV) or C (HCV) inducessevere liver cirrhosis and inflammation as seen in more than 80%of HCC patients (El-Serag, 2012). Like the papillomaviruses, thesetwo hepatitis viruses have carcinogenic properties that act on keycellular targets involved in apoptosis and promotion of the cellcycle during cancer progression (reviewed in: McGivern andLemon, 2011; Fallot et al., 2012; Arzumanyan et al., 2013). Bothhepatitis viruses have also been studied for their effect on theHIF-1 pathway and its role in HCC progression. As stated previously,HIF-1 activity can be induced through disruption to its oxygen-dependent degradation, as well as by the oxygen-independentincrease in growth factor signaling. The latter mechanism appearsto occur in HBV-induced HCCs.

The major factor in the development of HCC induced by HBVis the HBx gene (Kim et al., 1991; Benn and Schneider, 1995; Fallotet al., 2012). In addition to inflicting genetic instability anddisrupting normal cell cycle control, the HBx protein (expressedby inducible Chang liver cells) independently increases the trans-lation of HIF-1α mRNA leading to an increase in its transcriptionalactivity on target genes (Yoo et al., 2003). This HIF-1α activation byHBx occurs via increased signaling of the MAPK pathway. Althoughthe study provided a qualitative assessment of HBx protein byTa

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S. Cuninghame et al. / Virology 456-457 (2014) 370–383374

western blot, quantification was not performed. Measurement ofinduced HBx mRNA expression was also not reported. In additionto enhancing HIF-1α synthesis, its stabilization was also increasedin HBx transfected liver and non-liver cells, by introducing terminaltruncations to HBx to determine the specific binding region. Thisstabilization was accomplished solely by the C-terminus of HBxdirectly binding to the C-terminus to HIF-1α, potentially interferingwith its association with PHDs or VHL (Yoo et al., 2004). A concurrent,but separate study found direct binding between full length HBx andHIF-1α leading to stabilization of HIF-1α by inhibiting its interactionwith VHL (Moon et al., 2004).

HBx can also indirectly cause a rise in HIF-1 via a signalingpathway containing the growth factor receptor, human epidermalgrowth factor receptor-2 (HER2, also known as ErbB2). Retroviraltransduction of the HepG2 cell line with HBx leads to a transcrip-tional upregulation of ERBB2, and corresponding increase in itsprotein level (Liu et al., 2009). Enhanced ERBB2 signaling thenleads to higher HIF-1α levels through enhanced protein synthesisvia the PI3K/Akt pathway (Laughner et al., 2001). Increased Aktphosphorylation has been reported in HBx transduced HepG2cells, potentially leading to downstream enhancement of HIF-1αsynthesis, although in this paper, the authors did not report dataon HIF-1α activity (Liu et al., 2009). Collectively, these studiesimply that the HBx protein increases HIF-1α activity through bothinhibition of its oxygen-dependent degradation pathway, as wellas through amplification of its synthesis through the MAPK path-way. Lastly, HBx has also been implicated in the upregulation ofHIF-1 signaling by inducing the transcription of HDAC1 andmetastasis-associated protein 1 (MTA1) leading to an increase inprotein levels in vitro and in vivo (liver tissue of HBx-transgenicmice), which stabilizes HIF-1 under hypoxic conditions by inhibit-ing its association with PHDs and VHL (Yoo et al., 2008). Whilethese results at first glance appear to be in conflict with thoseinvolving HPV E7 and HDACs, it is possible that enhancementof HDAC1 transcription by HBx inhibits HIF-1α degradation,while binding of HPV E7 to HDACs augments its ability toform transcriptional complexes—this remains to be confirmed

experimentally. Thus, the key oncogenic protein of HBV hasacquired several mechanisms leading to upregulation of HIF-1α,which likely accounts for its significant association with thedevelopment and progression of HCCs.

Hepatitis C virus has also independently acquired its ownmechanisms to increase HIF-1α. The first evidence of HIF-1αmodulation by HCV came in 2007 when Nasimuzzaman andcolleagues showed that HCV's non-structural proteins were cap-able of inducing HIF-1α protein levels, even under normoxicconditions (Nasimuzzaman et al., 2007). Mechanistically, HCVinfection induces oxidative stress, which in turn, activates severalkinase pathways (Wang et al., 1998), including the PI3K and MEKpathways that lead to increased HIF-1α synthesis (Tardif et al.,2005; Nasimuzzaman et al., 2007). Treatment of HCV-infected HCCHuh-7 cells with MEK and PI3K inhibitors was sufficient tocompletely block the induction of HIF-1α. Although the authorsused a liver-derived cell line, it would have been interesting toobserve the effect of HCV-infection on normal hepatocytes, sinceHCC Huh-7 cells may already have altered pathways connected toHIF-1. In addition to the non-structural proteins, the HCV Coreprotein can independently induce HIF-1α and promote HCC (Rayet al., 1997; Moriya et al., 1998). Under hypoxic conditions, stabletransfection with the HCV Core protein (but not the non-structuralprotein NS5A or an empty vector) augmented HIF-1α by tran-scriptionally upregulating HIF-1α mRNA via activation of theNF-kB signaling pathway through an undetermined mechanism(Abe et al., 2012). However, these results were found in HAK-1Ahepatoma cells already containing HCV, so it is unclear howrelevant this overexpression of Core is in relation to a naturalHCV infection.

The oncogenic herpes viruses

Of the nine distinct human herpes virus (HHV) types knownto cause disease, only the γ-herpes viruses—HHV-4 (Epstein–Barrvirus or EBV) and HHV-8 (Kaposi's sarcoma-associated herpesvirus or KSHV)—are oncogenic, and both are frequently found to

Fig. 2. Oncoviruses function to enhance HIF-1 transcriptional activity. Infection with an oncovirus in its respective target cells allows for the expression of specificoncoproteins that lead to HIF-1α accumulation, followed by translocation to the nucleus. Here, it forms a complex with HIF-1β and p300/CBP that can then bind to HRE’s inthe promoters of and amplifies their expression. Viral proteins are color coded for their respective virus shown at top: HPV (red), HCV (yellow), HBV (blue), EBV (green),KSHV (pink), HTLV-1 (teal). Refer to abbreviation list for full names. A rectangular border represents nucleic acids and ellipses are proteins. Black and white backgroundsrepresent specific HIF-1 and effector cellular components, respectively. The dotted line represents the nuclear membrane.

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cause cancers in immunocompromised individuals (Ramos daSilva and Elgui de Oliveira, 2011). EBV infections promote severalcancers, including Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, and nasopharyngeal carcinoma (Thompsonand Kurzrock, 2004). KSHV is a causative agent of Kaposi’ssarcoma and primary effusion lymphoma—two cancers that areassociated with HIV infection (Wen and Damania, 2010). Each ofthese two oncogenic herpes viruses are also capable of increasingHIF-1α levels.

EBV primarily infects B cells of the immune system and cantransform them to latently infected lymphoblastoid cell lines (LCL)in vitro. This occurs upon expression of a distinct repertoire oflatent viral proteins known as the EBV nuclear antigens (EBNAs)and latent membrane proteins (LMPs) (Young and Rickinson,2004; Kalla and Hammerschmidt, 2012). EBV’s contribution toHIF-1 activity was first noted when LCLs of Type II and Type IIIlatency expressing LMP-1 showed increased HIF-1α protein levelsover HeLa control cells under normoxic conditions (Wakisakaet al., 2004). Furthermore, stable or transient transfection of thenasopharyngeal carcinoma cell line Ad-AH with LMP-1 lead to asignificant increase in HIF-1α protein levels as well as nucleartranslocation and transcriptional activity as measured by a lucifer-ase assay of the VEGF promoter containing an HRE. The underlyingmechanism of LMP-1-induced HIF-1α protein level increase wasdetermined to be solely due to an increase in its mRNA translation,rather than transcription, by activation of the p42/p44 MAPKpathway, but not of the p38 MAPK pathway (Wakisaka et al., 2004).While the classical MEK/MAPK pathway is typically associated withpromoting cell division and proliferation in response to growthfactor stimulation, the p38/MAPK pathway is known, along withthe JNK/MAPK pathway, to respond to inflammatory stressesand elicit apoptosis and cell cycle arrest in a variety of cell types(for review of mammalian MAPK pathways, see Qi and Elion,2005). In LMP-1-transfected Ad-AH cells, HIF-1α accumulationoccurs via LMP-1’s effect on increasing production of reactiveoxygen species (ROS), particularly H2O2. This oxidative stressactivates MAPK signaling (Chandel et al., 2000; Son et al., 2011).Increased signaling along the p42/p44 MAPK pathways upon EBVLMP-1 transfection leading to HIF-1 activation could be the resultof a direct LMP-1-induced increase in H2O2 levels. In addition toenhancing the synthesis of HIF-1α, the same group later foundthat LMP-1 also functions to inhibit the degradation of HIF-1α byincreasing the level of Siah-1 protein, a key E3 ubiquitin ligase thatcan induce the degradation of the PHDs required for HIF-1αbreakdown (Kondo et al., 2006). LMP-1 activation of Siah-1 wasregulated at the protein level through inhibiting its breakdown.However, the precise mechanism of inhibiting Siah-1 breakdownby LMP-1 was not determined. This is the first evidence of anoncogenic viral protein interfering with the oxygen-dependentdegradation of HIF-1α by inhibiting PHD activity, and provides anovel mechanism for HIF-1 activation to be explored in othercancers, particularly those of exogenous origin.

Several nuclear antigen proteins of EBV also function toincrease HIF-1α. EBNA-1 enhances the transcriptional activity ofactivator protein 1 (AP-1), a transcription factor that targets theHIF-1α gene. Evidently, stable transfection of EBNA-1 into Ad-AHcells acts to initiate the transcription of HIF-1α (O’Neil et al., 2008).However, whether the enhanced transcription of HIF-1α wasmediated solely by the AP-1 transcription factor was not deter-mined. Two other nuclear antigen proteins act to interfere withthe degradation pathway of HIF-1α. As described previously, theoxygen-dependent degradation of HIF-1α requires the activity ofPHD-1, PHD-2, and PHD-3 to hydroxylate HIF-1α, which is thenubiquitinylated by VHL and degraded by the 26S proteasome.Recent work illustrates that infection of full length EBV increasesHIF-1α protein levels and translocation to the nucleus in

comparison to normal cytokine-induced proliferating B cells.Levels of HIF-1α mRNA, however, were in fact lower in newlyinfected EBV cells and LCL’s in comparison to cytokine-inducedproliferating B cells, indicating that HIF-1α protein expressionalterations were post-transcriptional and that EBV infectiondirectly results in an HIF-1α increase, rather than resulting fromproliferation (Darekar et al., 2012). Two EBV nuclear antigenproteins, EBNA-3 and EBNA-5, bind directly to PHD-2 and PHD-1,respectively. In glutathione S-transferase pull-down assays of LCLsbinding of the EBV nuclear antigens to their appropriate PHDbinding partner leads to a concomitant increase in HIF-1α levelswithin the nucleus, where it can form a heterodimer with HIF-1βand activates the transcription of several target genes.

The second of the two oncogenic herpes viruses, KSHV, causesKaposi’s sarcoma, a cancer highly prevalent in immunosuppressedindividuals (Mesri et al., 2010). The genome of KSHV is relativelylarge in comparison to other oncogenic viruses (e.g., approximately170 kilobase pairs for KSHV vs. 8 kilobase pairs for HPV), andcontains over 80 open reading frames (ORFs) (Russo et al., 1996).The 74th ORF of the genome encodes a KSHV viral G-proteincoupled receptor (vGPCR) (Bais et al., 1998, 2003; Montaner et al.,2001). Stable transfection of vGPCR into NIH 3T3 mouse fibroblastcells results in activation of MEK and p38 signaling cascades,leading to direct phosphorylation of HIF-1α, and thus subsequentincreases in HIF-1 transcriptional activity (Sodhi et al., 2000). Theactivation of these two MAPK pathways is diminished in a dose-dependent manner by MEK/MAPK and MMK6/p38 pharmacologi-cal inhibitors in the vGPCR cells, with a correlative decrease intranscription of HIF-1 target genes. While this study shows thatvGPCR, a report on the level of vGPCR mRNA or protein was notincluded. As well, given the shear number of ORFs in KSHV,studying vGPCR alone may be missing the full complexity of KSHVand its affect on the HIF-1 pathway. In a separate study, vGPCR-expressing cells were found to secrete cytokines that act onneighboring cells in a paracrine fashion to induce HIF-1 and HIF-2 through the Akt, ERK, p38, and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) pathways, that in turn enhancemTOR-mediated HIF upregulation (Jham et al., 2011). Blockingthe synthesis of HIF-1α using digoxin inhibited the in vivo tumori-genic ability of vGPCR, again indicating that the viral activation ofHIF-1 enhances carcinogenesis. Furthermore, the latent nuclearantigen-2 (LANA-2) gene of KSHV, also known as viral interferonregulatory factor 3 (vIRF-3), is implicated in the stabilization ofHIF-1α, in addition to its oncogenic role of p53 inhibition (Rivaset al., 2001). While no differences in mRNA levels were reported,HIF-1α stability was enhanced by LANA-2/vIRF-3 when trans-fected in SLK endothelial cells (Shin et al., 2008). Under normoxicconditions, LANA-2/vIRF-3 binds to the bHLH domain of HIF-1αand inhibits the breakdown of HIF-1α, which seemingly does nothave an impact on its dimerization capability, and only furtherenhances the nuclear localization and transcriptional activity ofHIF-1. Although it is now known that LANA-2/vIRF-3 stabilizesHIF-1α leading to its increased transcriptional activity, theexact mechanism of how this occurs remains to be elucidated.Additionally, considering vIRF3 is specifically expressed in host Bcells and associated with primary effusion lymphoma (PEL) (Rivaset al., 2001), its effect on the HIF-1 pathway in endothelial cellsmay not be congruent in a B cell environment.

The LANA-1 protein of KSHV plays a key role in KSHV’soncogenicity, as it has pRb inhibiting capacity coincident withthe ability to obstruct p53 activity (Friborg et al., 1999; Radkovet al., 2000). Like LANA-2/vIRF-3 and vGPCR, which can enhanceHIF-1 activity under normoxic conditions, LANA-1 also has HIF-1regulating properties. While LANA-2/vIRF-3 increases HIF-1αactivity by binding directly to it, LANA-1 further augments HIF-1α stabilization in KSHV-transfected B cells by degrading VHL in

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the EC5S ubiquitin complex (Cai et al., 2006a). While cotransfec-tion experiments showed HIF-1α to be enhanced by LANA-1, nocomment on endogenous levels of HIF-1 was made until a followup study where the same group found that HIF-1α proteinlevels are higher in KSHV-positive PEL lines in comparison toKSHV-negative cell lines, and LANA-1 stimulates the nuclearaccumulation of HIF-1α (Cai et al., 2007). RNA interference wasused to show that a decrease in LANA-1 was coincident with adecrease in HIF-1α protein levels. The specific regions required forthe enhanced nuclear translocation of HIF-1α by LANA-1 weredetermined to be their oxygen-dependent domain (ODD), andthe N-terminus amino acids 46–89, respectively. Lastly, thereappears to be a role for KSHV in enhancing the transcription ofHIF-1α mRNA in endothelial cells (Carroll et al., 2006). Clearly, theKSHV virus has adopted numerous distinct mechanisms thatactivate hypoxic signaling in infected cells, even under normoxicconditions.

Interestingly, promoters of key KSHV genes have been foundto contain HREs (Haque et al., 2003), and activation of HIF-1αtranscriptional activity by LANA-1 leads to expression of theselytic-reactivating genes (Cai et al., 2006b). It appears that LANA-1,LANA-2/vIRF-3, and vGPCR enhance HIF1 activity, which in turninduces lytic KSHV genes containing HREs, transitioning the virusfrom its latent to lytic replication stage.

The human T-cell lymphotropic virus

The human T-cell lymphotropic virus-1 (HTLV-1) is the onlyretrovirus known to be capable of directly causing cancer (adultT-cell lymphomas or leukemias) in humans (Moore and Chang,2010). The HTLV-1 Tax protein is critical for the virus’ ability totransform and immortalize T cells (Grassmann et al., 2005). TheTax protein is responsible for the activation of several oncogenicsignaling pathways, including the PI3K/Akt pathway (Peloponeseand Jeang, 2006; Jeong et al., 2008). In a robust study by Tomitaand colleagues, HIF-1α protein levels were increased even undernormoxic conditions in HTLV-1 positive cell lines in comparison toproliferating T-cell leukemia cell lines not infected with HTLV-1due to Tax’s effect of enhancing PI3K/Akt signaling (Tomita et al.,2007). Mutants of Tax that were unable to activate PI3K/Aktshowed HIF-1 transcriptional activity comparable with untrans-fected controls. Pharmacological inhibition of the PI3K/Akt path-way showed a dose-dependent decrease in HIF-1 activity seenupon wild-type Tax transfection. Levels of HIF-1α mRNA levelswere found to be comparable between both uninfected and HTLV-infected leukemia lines, meaning that the augmentation of HIF-1αprotein levels lies solely in post-transcriptional alterations. Theproliferation of these cell lines appear directly affected by Tax-induced HIF-1 activity, as knockdown of HIF-1α by way of siRNAsignificantly decreased the growth rate of HTLV-1 infected leuke-mia cell lines. This is yet another confirmation that activation ofHIF-1α by oncogenic viruses is a key pathway conferring enhancedproliferation, likely by targeting key downstream targets involvedin cancer progression.

Downstream activation of genes involved in virally-inducedcancer progression by HIF-1

Metabolism

In normal, differentiated cells, glucose is metabolized to pyr-uvate, followed by oxidative phosphorylation in order to maximizeadenosine triphosphate (ATP) as a cellular energy source, howevercancer cells exhibit glucose metabolism resembling that of anae-robic cells (Vander Heiden et al., 2009). Under aerobic conditions,

pyruvate is converted to acetyl-CoA, which subsequently entersthe mitochondrial tricarboxylic acid (TCA) cycle, the products ofwhich (NADH, FADH2) donate electrons to the protein complexesof the electron transport chain, generating a proton gradientacross the inner mitochondrial membrane. Relying on the pre-sence of molecular oxygen as a final electron acceptor, protons aresubsequently transported from the inter-membrane space into themitochondrial matrix via ATP synthase, which synthesizes ATPfrom ADP and inorganic phosphate (Schultz and Chan, 2001).Under hypoxic conditions, however, molecular oxygen is notavailable as a final electron acceptor, so pyruvate is not convertedto acetyl-CoA and subsequent oxidative phosphorylation does notoccur. Rather, pyruvate is converted to lactate by the ubiquitousenzyme, lactate dehydrogenase, a process largely governed byactivation of HIF-1 and subsequent adaptive gene expression(Semenza, 2000). It is precisely this hypoxic-like metabolism,with lactate as the major byproduct, that cancer cells perform,even in the presence of adequate oxygen—this phenomenon wasfirst described by famed German physiologist, Otto Warburg, in the1920s and thus termed theWarburg effect (Warburg et al., 1927; Shaw,2006). It is generally thought that the Warburg effect is a requirementfor immortalization andmalignant transformation of cells, likely due tothe reduction in senescence-inducing ROS (e.g., H2O2, O2

�) that arisefrom a primarily glycolytic mechanism of ATP production, as well aspermitting adaptation to the hypoxic environment typical of tumors(Brand and Hermfisse, 1997; Kondoh et al., 2005; Kim and Dang, 2006;Dolado et al., 2007).

Recent evidence has shown that upon infection of certain hosttarget cells, oncoviruses induce molecular changes that account fora Warburg effect (Noch and Khalili, 2012). In some instances, thesemetabolic changes are induced following acute infection with anoncovirus (i.e., prior to the formation of a detectable tumor).Therefore, it is likely that the induction of the Warburg effect,either directly or indirectly, confers a proliferative advantage forinfected cells and is a necessary step to cancer. Among the genesthat are activated by HIF-1 and involve the Warburg effect arethose constituting a transition from oxidative phosphorylationto predominately aerobic glycolysis. Metabolic gene targets ofHIF-1 have been known for years and it is clear that the enzymescatalyzing glucose metabolism—phosphoglycerate kinase 1 (PGK1),hexokinase 1 and 2 (HK1/2), glyceraldehyde-3-phosphate dehydro-genase (GAPDH), phosphofructokinase (PFK), and the glucose trans-porters (GLUT-1, GLUT-3)—are upregulated by HIF-1 (Iyer et al., 1998).Furthermore, key enzymes that contribute to the Warburg effect,such as PDK1 and LDHA, are also HIF-1 targets (Semenza et al.,1994; Lu et al., 2002; Kim and Dang, 2006; Semenza, 2007).

Due to HIF-1’s effect on metabolism, it is not surprising that anincrease in lactate production (i.e., Warburg effect) coincides withHIF-1α upregulation by oncoviruses. Early histopathological stu-dies of HPV16 transgenic mice reported a concomitant increase inthe mRNA levels of GLUT-1, PGK-1, and HIF-1α in basal keratino-cytes during epidermal carcinogenesis (Elson et al., 2000). Simi-larly, in EBV-infected LCL cells, EBNA proteins that bind to key PHDproteins (involved in HIF-1α’s oxygen dependent degradation)showed a significant increase in metabolic enzymes (approxi-mately 5, 4.5, 3.25, 6.5, and 2 fold increase for PGK1, GAPDH,LDHA, GLUT-1, PDK1, respectively), along with an increase inaerobic glycolysis (i.e., pyruvate and lactate acid production)(Darekar et al., 2012). HCV viral protein expression has beenshown to increase HIF-1α levels in osteosarcoma cells transfectedwith HCV genes, as well as the transcription of glycolytic enzymegenes and other HIF-1α target genes. These observations coincidewith a decrease in oxidative metabolism, forcing ATP productionto occur largely via non-oxidative glycolytic metabolism, a HIF-1controlled 2–2.5-fold transcriptional increase of the glycolyticgenes, LDHA and HK1 (Ripoli et al., 2010). These observations

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translate in vivo, where biopsies of HCV-positive hepatocellularcarcinomas show a significant increase in HIF-1α and HK1 mRNAover controls. Levels of LDHA mRNA were found to be higher aswell, albeit not significantly (Ripoli et al., 2010). These in vivoresults are in agreement with recent studies showing that HCVCore protein transcriptionally upregulates HIF-1α mRNA (Abeet al., 2012). Other independent studies on the temporal proteomeof HCV-infected Huh-7.5 cells found changes consistent with ashift from oxidative metabolism toward aerobic glycolysis, asevidenced by increases in several metabolic HIF-1 targets thatare markers of the Warburg Effect upon acute HCV infection(Diamond et al., 2010). Whether these increases in marker proteinlevels correlate with a biologically relevant alteration in glucosemetabolism by HIF-1 remains to be determined.

In discussions of tumor metabolism, two schools of thoughtexist. Some contend that HIF-1 induction of the Warburg effect incancer cells is simply the result of the hypoxic tumor microenvir-onment (Garber, 2004). Others argue that the activation of theWarburg effect by HIF-1 is an intrinsic mechanism cancer cellshave in order to ensure survival and continued proliferation uponexposure to impending tumor hypoxia, and that without thepreceding activation of HIF-1, tumor formation would not befeasible (Garber, 2004). Central to the latter contention is the factthat many pathways (e.g., the PI3K & MAPK pathways) that are oftendysregulated in cancer have an impact on both HIF-1 activity andother pathways involved in cancer progression. As described above,cancers of exogenous origin have (often several) hard-wired mechan-isms to increase HIF-1α levels. This supports the notion that HIF-1 isan important factor in permitting initial tumor formation, perhaps byallowing for adaptation to the ensuing hypoxic microenvironmenttypical of most solid tumors. Some of these HIF-1-inducing oncov-iruses cause systemic cancers (e.g., EBV and HTLV-1 induction ofB- and T-cell lymphomas, respectively). In such cases where environ-mental hypoxia in tumor formation is not a characteristic, HIF-1 islikely a key player in the malignant transformation pathway, and notsimply a consequence of hypoxia.

Angiogenesis

As with normal tissues, tumors require a constant supply ofnutrients that are obtained through the vasculature to ensure cellsurvival and permit proliferation. To ensure these demands of therapidly proliferating cells are met, tumors have mechanisms toensure the formation of blood vessels through angiogenesis(Hanahan and Weinberg, 2011; Carmeliet and Jain, 2011). Newblood vessels in the microenvironment are formed by enhancingpro-angiogenic or by inhibiting anti-angiogenic factors. Perhapsthe most studied of the tumor angiogenic factors are those of thevascular endothelial growth factor (VEGF) family, which is also atranscriptional target of HIF-1 (Forsythe et al., 1996). In fact, wehave already discussed several studies containing reports of HIF-1transcriptional activity based on its ability to activate VEGF-Aexpression that, in turn, induces angiogenesis via the VEGFreceptor-2 (Carmeliet and Jain, 2011). In this section, we will focuson each oncogenic virus’ contribution to angiogenesis via HIF-1.

The oncogenes of HPV have been examined for their ability toinduce VEGF expression and angiogenesis via the HIF-1 pathway.The in vitro activation of HIF-1α via E6-induced p53 degradationsignificantly increases expression of a VEGF reporter gene contain-ing the promoter region of VEGF (which also contains a HIF-1 HRE)(Ravi et al., 2000). Additionally, in non-small cell lung cancer cells,the overexpression of both HPV16 E6 and E7 oncoproteins isassociated with the promotion of angiogenesis both in vitro andin vivo, and is due to enhanced HIF-1α, as well as downstreamVEGF mRNA and protein expression (Li et al., 2011). These studiesalign with others that have found high-risk HPV oncoproteins

capable of inducing pro-angiogenic factors that are transcriptionaltargets of HIF-1 (Le Buanec et al., 1999; Bequet-Romero and López-Ocejo, 2000; López-Ocejo et al., 2000). Not surprisingly, pathologystudies reveal a similar trend—expression of multiple VEGF isoformsare significantly increased coincidently with the grade of cervicalintraepithelial neoplasia (Branca et al., 2006; Baritaki et al., 2007).These reports agree with the mechanistic findings previouslydescribed, wherein both E6 and E7 oncoproteins of high-risk HPVhave been found to directly activate HIF-1. Thus, E6 and E7 are likelymajor factors contributing to VEGF induction, in addition to thedevelopment of a hypoxic tumor microenvironment.

Angiogenic factors have also been studied in the context ofhepatitis viruses. Both HCV and HBV upregulate VEGF expressionthrough HIF-1. In the case of HCV-positive HCC, several markers ofangiogenesis have been found in vivo to be upregulated, notablyVEGF-A (Salcedo et al., 2005; Mas et al., 2007). Moreover, VEGF-Awas found to be expressed to significantly higher levels in HCV-HCC than in pre-malignant HCV-induced liver cirrhosis, due toVEGF’s importance in cancer development and progression.Experimental in vitro studies have confirmed these findings: VEGFexpression follows the increase of HIF-1α that occurs fromenhanced HIF-1α synthesis via oxygen-independent pathways(Nasimuzzaman et al., 2007). Soon after reports of HBV increasingboth HIF-1α translation via the MAPK pathway and VEGF angio-genic signaling (Lee et al., 2000), it was also revealed that HBxbinds to HIF-1α and induces VEGF expression (Moon et al., 2004).Recent work has implicated the mTOR pathway in VEGF produc-tion in HCC by induction of the inflammatory tumor necrosisfactor (TNF)-α signaling pathway. A downstream kinase of theTNF-α pathway, the β subunit of the IKKβ, activates mTORsignaling via repression of the TSC1/TSC2 tumor suppressorcomplex, which in turn leads to enhanced angiogenesis (Leeet al., 2007). Whether or not the mechanism of IKKβ-inducedangiogenesis is HIF-1-mediated VEGF transcriptional upregulationremains to be determined, but it is highly likely to at least be acontributing factor, as enhanced mTOR signaling clearly augmentsHIF-1 protein synthesis (Majumder et al., 2004; Thomas et al.,2005; Demidenko and Blagosklonny, 2011), and environmentalhypoxia has shown a similar effect on VEGF signaling via mTOR(Humar et al., 2002). Interestingly, the HBx oncoprotein alsoactivates mTOR signaling and VEGF-A angiogenesis through induc-tion of IKKβ, and activation of key modulators in this pathway iscorrelated with poor prognosis (Yen et al., 2012). Again, it remainsto be determined if this activation of angiogenic induction requiresHIF-1. The exact mechanism of IKKβ upregulation by HBx remainsto be elucidated but represents a possible novel pathway ofangiogenic signaling (potentially through HIF-1 induction) incancers arising from viral infection.

Given the promotion of angiogenesis by papillomaviruses andhepatitis viruses through HIF-1, it is not surprising that EBV andKSHV also induce VEGF expression in a similar manner. Severalstudies of EBV viral proteins that contribute to HIF-1 induction haveincluded analyses of HIF-1-mediated VEGF upregulation. Followingearly studies that alluded to EBV enhancing VEGF expression(Murono et al., 2001; Krishna et al., 2006; Hui et al., 2002), eachof LMP-1 (Wakisaka et al., 2004) and EBNA-1 (O’Neil et al., 2008),when studied for their HIF-1 induction capacity, presentedincreases in VEGF production. Similarly, while trying to determinethe molecular underpinnings and the importance of angiogenesisin Kaposi’s sarcoma (Cornali et al., 1996; Masood et al., 1997; Baiset al., 1998), HIF-1α was shown to be activated by KSHV’s vGPCRby pathways acting on the mTOR complex that lead to VEGFproduction. In separate studies, the MAPK and p38 pathways(Sodhi et al., 2000), as well the paracrine activation of Akt, ERK,p38 and IKKβ on mTOR (Jham et al., 2011) result in VEGF inductionby this oncogenic virus. In studies using VEGF itself or its promoter

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(as a proxy of HIF-1 activity), LANA-1 (Cai et al., 2007) and LANA-2/vIRF-3 (Shin et al., 2008), through the induction of HIF-1, lead totranscriptional accumulation of VEGF. Lastly, these results aremirrored with regard to human T-cell lymphotropic virus, whereHIF-1α levels are increased via activation of the PI3K/Akt pathway,with a concomitant increase in VEGF mRNA (Tomita et al., 2007).

Invasion and metastasis

It is well documented that metastasis is responsible for up to90% of cancer deaths, however, researchers are still unraveling thecomplex mechanisms that contribute to its promotion. Following thediscovery that HIF-1 activation is common in cancer (Zhong et al.,1999), it was promptly shown that it not only contributes to metabolicreprogramming and angiogenesis, but also to metastatic potential bytranscriptionally activating several genes that promote invasiveness(Krishnamachary et al., 2003; Semenza, 2003; Fujiwara et al., 2007).Consequently, several of the key genes involved in amplifying meta-static potential have also been found to be upregulated in both solidand systemic malignancies of viral origin by altering cell adhesion andmotility as well as the tumor microenvironment.

Cell adhesion and motilityThe downregulation of the epithelial adhesion molecule, E-cad-

herin, is a common characteristic of carcinomas that promotesinvasion (Schipper et al., 1991; Oka et al., 1993; Umbas et al., 1994;Onder et al., 2008; Ma et al., 2010). E-cadherin repression allowsfor epithelial–mesenchymal transition (EMT)—an event requiredfor proper embryonic development but also commonly observedwith carcinoma metastasis (Chua et al., 2006; Christiansen andRajasekaran, 2006; Acloque et al., 2008). Hypoxia can act as astressor for EMT activation via HIF-1 (Higgins et al., 2007), becauseseveral genes that are E-cadherin repressors and involved inembryonic development, such as Snail and Twist, are HIF-1 targets(reviewed in Yang and Wu, 2008). A comprehensive study on HCCfound a significant correlation of Snail and Twist induction inpromoting EMT and metastasis, particularly for HBV- and HCV-positive samples (Yang et al., 2009). Following this report, in vitrostudies indicated that HCV glycoproteins E1 and E2 can contributeto cancer cell migration through the upregulation of Snail andTwist, by virtue of an HIF-1-dependent mechanism (Wilson et al.,2012). Similarly, the LMP-1 protein of EBV that can enhance HIF-1α levels via mTOR signaling (Wakisaka et al., 2004) also increasesTwist (Horikawa et al., 2007) and Snail (Horikawa et al., 2011)expression. Each of these studies found the upregulation of Snailand Twist to inversely correlate with E-cadherin levels andassociate with clinical metastases for nasopharyngeal carcinoma(Horikawa et al., 2007, 2011). Following reports of HPV16 E6’sability to inhibit E-cadherin levels (Matthews et al., 2003), similarresults to those of hepatitis and Epstein–Barr viruses have beenfound, where Snail, Twist and/or other E-cadherin repressors thatare targets of HIF-1 are upregulated by infection with HPV (Leeet al., 2008; D’Costa et al., 2012). Complementary findings havebeen reported for KSHV as well, where an endothelial-to-mesenchymal transition is observed (Gasperini et al., 2012).Whether or not these results are, in part, mitigated by theirrespective viral oncoproteins’ effect on HIF-1 described previouslyremains to be determined.

The microenvironmentResearch over the past few decades has highlighted the

importance of the tumor microenvironment and its associatedcomponents in promoting invasion into the bloodstream andsubsequent colonization at distant sites. These metastatic-promoting factors may originate from tumor cells themselves or

from the very cells of the tumor microenvironment itself (e.g.,tumor-associated macrophages) (environmental factors drivingcancer cell metastasis reviewed in: Steeg, 2006; Joyce and Pollard,2009). One particular environmental factor driving cancer cellmetastasis and influenced by HIF-1 is pH. The HIF-1-induced War-burg effect results in a significant increase in lactic acid production,and thus, a requirement for tumor cells to combat a decrease in pH inorder to survive. It is not surprising then, that HIF-1 upregulatesseveral pH-regulating membrane transporters, such as a lactatetransporter (monocarboxylate transporter 4) and carbonic anhydraseIX and XII (CAIX/XII) (Parks et al., 2013). While CAIX is often simplyused as a hypoxic marker, it also correlates with advanced cancerprogression and poor overall survival (Bui et al., 2003; Swinson et al.,2003; Potter and Harris, 2004). Of particular interest is CAIX’s role incounteracting intracellular tumor acidosis (i.e., Warburg effect) bycatalyzing the reversible hydration of carbon dioxide, accounting forreports of an observable decrease in extracellular pH, while con-tributing to tumor growth (Švastová et al., 2004; Chiche et al., 2009).Recent work has provided evidence of enhanced cancer metastasisunder acidic conditions and tumor growth being directed to sites oflow pH, indicating that the acidic microenvironment is a key aspectof extracellular matrix remodeling (Estrella et al., 2013). Furthermore,treatment with NaHCO3 as pH buffer significantly increases the pH ofthe tumor microenvironment, while decreasing metastases (Robeyet al., 2009; Estrella et al., 2013). These results may explain additionalroles of HIF-1 in enhancing tumor metastases and invasion, via theupregulation of CAIX.

Several tumor viruses have been documented to enhance CAIXexpression, often concomitantly with HIF-1 activity increases.The cervical carcinoma cell lines, SiHa and CaSki, which harborintegrated HPV16, have been found to overexpress CAIX (Lee et al.,2008). CAIX expression is further augmented (Niccoli et al., 2012)by aggressive HPV16 E6 variants, such that the Asian-American E6variant (Zehbe et al., 1998), which is found eight times more oftenin invasive cervical cancer than prototype E6, relative to non-cancer controls (Berumen et al., 2001). Thus, CAIX likely representsa key protein in tumor microenvironment hypoxia, and in promot-ing invasion. Similar results have been reported for the HBxprotein of HBV: transient transfection of Hep G2 cells with HBxleads to an increase in CAIX mRNA expression and protein level viaHIF-1 (Holotnakova et al., 2010). Clinical specimens of nasophar-yngeal carcinoma, an epithelial cancer for which infection withEBV is a significant risk factor (Lo et al., 1999; zur Hausen et al.,1970), have been found to exhibit increased expression of HIF-1αand CAIX, and the presence of the latter significantly correlatesnegatively with progression-free survival (Hui et al., 2002).

As mentioned, the upregulation of CAIX by HIF-1 is associatedwith a decrease in extracellular pH (Chiche et al., 2009), whichlikely provides an optimal environment of matrix remodelingproteases (such as cathepsins and metalloproteases), as theyhave been found to have highest activity in an acidic milieu(Buck et al., 1992; Turk et al., 2001; Rofstad et al., 2006). Matrixmetalloprotease-2 (MMP-2) and MMP-9 are two more of the manyHIF-1 targets involved in promotion of angiogenesis and invasionthrough proteolytic cleavage of extracellular matrix components(Semenza, 2003; Choi et al., 2011). Indeed, either one or both ofthese MMPs have been found to have increased expression and/oractivation of their respective zymogens by oncoviruses including,HPV16 (Coussens et al., 2000; Cardeal et al., 2006), EBV (Yoshizakiet al., 1998; Horikawa et al., 2000), HCV (Lichtinghagen et al.,2003; Núñez et al., 2004), HBV (Kuo et al., 2000; Chung et al.,2004; Liu et al., 2010), and KSHV (Wang et al., 2004; Qianet al., 2007). It is worth noting that few studies have reportedwhether or not activation of these MMPs was directly due to viralgenes’ activation of HIF-1—this remains a subject of futureexploration. However, some studies have documented that the

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role of the upstream signaling pathways that enhance HIF-1activity (described earlier) are required for activation of the MMPsunder investigation (Chung et al., 2004) and several of the viraloncogenes (such as LMP-1 of EBV) required for target cell trans-formation and HIF-1, also induce MMP expression (Wakisaka andPagano, 2003).

Several additional genes involved in the invasion and metastasiscascade, such as cathepsin D and urokinase plasminogen activatorreceptor, among others, are targets of HIF-1 (Semenza, 2003).Whether or not these and other genes involved in metastasis arealso activated by HIF-1 in oncoviral cancers has not yet been resolvedand remains an attractive area for future work, given the exhaustiveevidence for oncoviral HIF-1 activation.

Conclusion and future directions

Following the discovery by Gregg Semenza and colleagues inthe mid-1990s that HIF-1 is a key regulator of oxygen homeostasisand adaptation to hypoxia came a plethora of studies elucidatingits importance in a variety of health conditions, including cardio-vascular disease and cancer. With regard to cancer, HIF-1 is wellknown to transcriptionally activate genes involved in metabolism,angiogenesis and invasion/metastasis, as it is typically inducedwithin the tumour microenvironment that is notoriously hypoxic.Thus, HIF-1 and its associated mechanisms of upregulation repre-sent an attractive target in the fight against malignancies withdetrimental prognoses.

In this review, we have exemplified a unifying theme and themechanisms therein of viral oncogenes in the activation of HIF-1.Key downstream target genes of HIF-1, including those of meta-bolism and those that collectively promote proliferation andmetastases, have been reviewed in order to accentuate this path-way in viral carcinogenesis. Most of the oncoviruses that havebeen linked to human cancers have been clearly documented toincrease HIF-1 activity, which generally correlates with a poorprognosis. A recently classified oncogenic virus, Merkel cell poly-omavirus (MCV) (Feng et al., 2008), is associated with aggressivecarcinomas. Although one study has found inconsistent HIF-1αstaining in Merkel cell carcinomas (Fernández-Figueras et al.,2006), further investigation is required to determine if there is arelationship between MCV and the HIF-1 pathway. While few ofthe oncoviruses discussed herein have the same method ofenhancing HIF-1 levels, the end result is the same: downstreamactivation of cancer-promoting genes. Although we have high-lighted numerous studies that exemplify a link between viralgenes and HIF-1, future studies should employ quantitative meth-ods to elucidate molecular mechanisms as present in a naturalinfection. Appropriate target cell selection for respective viralinfections is important to fully understand the context of theseviral genes on HIF-1. Ultimately we require a thorough under-standing of how intrinsic host cell HIF-1 regulation is affected byviral genomes and their life cycles. However, given the exhaustiveevidence for HIF-1 activation by oncoviruses, HIF-1 or the viralgenes responsible for its activity represent ideal candidates fortargeted therapeutics for cancers of viral etiology.

Acknowledgments

This work was funded by a Natural Sciences and EngineeringResearch Council of Canada (NSERC) Grant to I.Z. (#435891-2013)and an NSERC Alexander Graham Bell Scholarship to S.C.(#442618-2013).

References

Abe, M., Koga, H., Yoshida, T., Masuda, H., Iwamoto, H., Sakata, M., Hanada, S.,Nakamura, T., Taniguchi, E., Kawaguchi, T., Yano, H., Torimura, T., Ueno, T., Sata,M., 2012. Hepatitis C virus core protein upregulates the expression of vascularendothelial growth factor via the nuclear factor-κB/hypoxia-inducible factor-1α axis under hypoxic conditions. Hepatol. Res. 42, 591–600.

Acloque, H., Thiery, J.P., Nieto, M.A., 2008. The physiology and pathology of the EMT.EMBO Rep. 9, 322–326.

Agani, F., Jiang, B.H., 2013. Oxygen-independent regulation of HIF-1: novel involve-ment of PI3K/AKT/mTOR pathway in cancer. Curr. Cancer Drug Targets 13,245–251.

Arzumanyan, A., Reis, H.M., Feitelson, M.A., 2013. Pathogenic mechanisms in HBV-and HCV-associated hepatocellular carcinoma. Nat. Rev. Cancer 13, 123–135.

Bais, C., Santomasso, B., Coso, O., Arvanitakis, L., Raaka, E.G., Gutkind, J.S., Asch, A.S.,Cesarman, E., Gerhengorn, M.C., Mesri, E.A., 1998. G-protein-coupled receptorof Kaposi’s sarcoma-associated herpesvirus is a viral oncogene and angiogen-esis activator. Nature 391, 86–89.

Bais, C., Van Geelen, A., Eroles, P., Mutlu, A., Chiozzini, C., Dias, S., Silverstein, R.L.,Rafii, S., Mesri, E.A., 2003. Kaposi’s sarcoma associated herpesvirus G protein-coupled receptor immortalizes human endothelial cells by activation of theVEGF receptor-2/KDR. Cancer Cell 3, 131–143.

Baritaki, S., Sifakis, S., Huerta-Yepez, S., Neonakis, I.K., Soufla, G., Bonavida, B.,Spandidos, D.A., 2007. Overexpression of VEGF and TGF-β1 mRNA in Papsmears correlates with progression of cervical intraepithelial neoplasia tocancer: implication of YY1 in cervical tumorigenesis and HPV infection. Int. J.Oncol. 31, 69–79.

Bersten, D.C., Sullivan, A.E., Peet, D.J., Whitelaw, M.L., 2013. bHLH-PAS proteins incancer. Nat. Rev. Cancer 13, 827–841.

Benn, J., Schneider, R.J., 1995. Hepatitis B virus HBx protein deregulates cell cyclecheckpoint controls. Proc. Natl. Acad. Sci. USA 92, 11215–11219.

BelAiba, R.S., Bonello, S., Zähringer, C., Schmidt, S., Hess, J., Kietzmann, T., Görlach,A., 2007. Hypoxia up-regulates hypoxia-inducible factor-1α transcription byinvolving phosphatidylinositol 3-kinase and nuclear factor κB in pulmonaryartery smooth muscle cells. Mol. Biol. Cell 18, 4691–4697.

Bequet-Romero, M., López-Ocejo, O., 2000. Angiogenesis modulators expression inculture cell lines positives for HPV-16 oncoproteins. Biochem. Biophys. Res.Commun. 277, 55–61.

Berumen, J., Ordoñez, R.M., Lazcano, E., Salmeron, J., Galvan, S.C., Estrada, R.A.,Yunes, E., Garcia-Carranca, A., Gonzalez-Lira, G., Madrigal-de la Campa, A.,2001. Asian-American variants of human papillomavirus 16 and risk for cervicalcancer: a case-control study. J. Natl. Cancer Inst. 93, 1325–1330.

Bodily, J.M., Mehta, K.P., Laimins, L.A., 2011. Human papillomavirus E7 enhanceshypoxia-inducible factor 1-mediated transcription by inhibiting binding ofhistone deacetylases. Cancer Res. 71, 1187–1195.

Boyer, S.N., Wazer, D.E., Band, V., 1996. E7 Protein of human papillomavirus 16induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res. 56, 4620–4624.

Branca, M., Giorgi, C., Santini, D., Di Bonito, L., Ciotti, M., Benedetto, A., Paba, A.,Costa, S., Bonifacio, D., Di Bonito, P., Accardi, L., Favalli, C., Syrjänen, K., 2006.Aberrant expression of VEGF-C is related to grade of cervical intraepithelialneoplasia (CIN) and high risk HPV, but does not predict virus clearance aftertreatment of CIN or prognosis of cervical cancer. J. Clin. Pathol. 59, 40–47.

Brand, K.A., Hermfisse, U., 1997. Aerobic glycolysis by proliferating cells: aprotective strategy against reactive oxygen species. FASEB J. 11, 388–395.

Bruick, R.K., McKnight, S.L., 2001. A conserved family of prolyl-4-hydroxylases thatmodify HIF. Science 294, 1337–1340.

Buck, M.R., Karustis, D.G., Day, N.A., Honn, K.V., Sloane, B.F., 1992. Degradation ofextracellular-matrix proteins by human cathepsin B from normal and tumourtissues. Biochem. J. 282, 273–278.

Bui, M.H., Seligson, D., Han, K.R., Pantuck, A.J., Dorey, F.J., Huang, Y., Horvath, S.,Leibovich, B.C., Chopra, S., Liao, S.-Y., Stanbridge, E., Lerman, M.I., Palotie, A.,Figlin, R.A., Belldegrun, A.S., 2003. Carbonic anhydrase IX is an independentpredictor of survival in advanced renal clear cell carcinoma: implications forprognosis and therapy. Clin. Cancer Res. 9, 802–811.

Cai, Q.L., Knight, J.S., Verma, S.C., Zald, P., Robertson, E.S., 2006a. EC5S ubiquitincomplex is recruited by KSHV latent antigen LANA for degradation of the VHLand p53 tumor suppressors. PLoS Pathog. 2, e116.

Cai, Q., Lan, K., Verma, S.C., Si, H., Lin, D., Robertson, E.S., 2006b. Kaposi’s sarcoma-associated herpesvirus latent protein LANA interacts with HIF-1α to upregulateRTA expression during hypoxia: latency control under low oxygen conditions.J. Virol. 80, 7965–7975.

Cai, Q., Murakami, M., Si, H., Robertson, E.S., 2007. A potential α-helix motif in theamino terminus of LANA encoded by Kaposi’s sarcoma-associated herpesvirusis critical for nuclear accumulation of HIF-1α in normoxia. J. Virol. 81,10413–10423.

Cardeal, L.B.D.S., Brohem, C.A., Corrêa, T.C.S., Winnischofer, S.M.B., Nakano, F.,Boccardo, E., Villa, L.L., Sogayar, M.C., Maria-Engler, S.S., 2006. Higher expres-sion and activity of metalloproteinases in human cervical carcinoma cell lines isassociated with HPV presence. Biochem. Cell Biol. 84, 713–719.

Carmeliet, P., Jain, R.K., 2011. Molecular mechanisms and clinical applications ofangiogenesis. Nature 473, 298–307.

Carroll, P.A., Kenerson, H.L., Yeung, R.S., Lagunoff, M., 2006. Latent Kaposi’ssarcoma-associated herpesvirus infection of endothelial cells activateshypoxia-induced factors. J. Virol. 80, 10802–10812.

S. Cuninghame et al. / Virology 456-457 (2014) 370–383380

Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez, J.A., Rodri-guez, A.M., Schumacker, P.T., 2000. Reactive oxygen species generated atmitochondrial complex III stabilize hypoxia-inducible factor-1α duringhypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275, 25130–25138.

Chiche, J., Ilc, K., Laferrière, J., Trottier, E., Dayan, F., Mazure, N.M., Brhaimi-Horn, C.,Pouysségur, J., 2009. Hypoxia-inducible carbonic anhydrase IX and XII promotetumor cell growth by counteracting acidosis through the regulation of theintracellular pH. Cancer Res. 69, 358–368.

Choi, J.Y., Jang, Y.S., Min, S.Y., Song, J.Y., 2011. Overexpression of MMP-9 andHIF-1α in breast cancer cells under hypoxic conditions. J. Breast Cancer 14,88–95.

Cheng, J., Kang, X., Zhang, S., Yeh, E.T., 2007. SUMO-specific protease 1 is essentialfor stabilization of HIF1alpha during hypoxia. Cell 131, 584–595.

Christiansen, J.J., Rajasekaran, A.K., 2006. Reassessing epithelial to mesenchymaltransition as a prerequisite for carcinoma invasion and metastasis. Cancer Res.66, 8319–8326.

Chua, H.L., Bhat-Nakshatri, P., Clare, S.E., Morimiya, A., Badve, S., Nakshatri, H., 2006.NF-κB represses E-cadherin expression and enhances epithelial to mesenchy-mal transition of mammary epithelial cells: potential involvement of ZEB-1 andZEB-2. Oncogene 26, 711–724.

Chung, T.W., Lee, Y.C., Kim, C.H., 2004. Hepatitis B viral HBx induces matrixmetalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential. FASEB J. 18, 1123–1125.

Cornali, E., Zietz, C., Benelli, R., Weninger, W., Masiello, L., Breier, G., Tschachler, E.,Albini, A., Stürzl, M., 1996. Vascular endothelial growth factor regulatesangiogenesis and vascular permeability in Kaposi’s sarcoma. Am. J. Pathol.149, 1851–1869.

Coussens, L.M., Tinkle, C.L., Hanahan, D., Werb, Z., 2000. MMP-9 supplied by bonemarrow-derived cells contributes to skin carcinogenesis. Cell 103, 481–490.

D’Costa, Z.J., Jolly, C., Androphy, E.J., Mercer, A., Matthews, C.M., Hibma, M.H., 2012.Transcriptional repression of E-cadherin by human papillomavirus type 16 E6.PloS One 7, e48954.

Darekar, S., Georgiou, K., Yurchenko, M., Yenamandra, S.P., Chachami, G., Simos, G.,Klein, G., Kashuba, E., 2012. Epstein–Barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent withthe Warburg effect. PloS One 7, e42072.

Depping, R., Steinhoff, A., Schindler, S.G., Freidrich, B., Fagerlund, R., Metzen, E.,Hartmann, E., Köhler, M., 2008. Nuclear translocation of hypoxia-induciblefactors (HIFs): Involvement of the classical α/β importin pathway. BBA – Mol.Cell. Res. 1783, 394–404.

de Martel, C., Ferlay, J., Franceschi, S., Vignat, J., Bray, F., Forman, D., Plummer, M.,2012. Global burden of cancers attributable to infections in 2008: a review andsynthetic analysis. Lancet Oncol. 13, 607–615.

Demidenko, Z.N., Blagosklonny, M.V., 2011. The purpose of the HIF-1/PHD feedbackloop: to limit mTOR-induced HIF-1α. Cell Cycle 10, 1557–1562.

Diamond, D.L., Syder, A.J., Jacobs, J.M., Sorensen, C.M., Walter, K.A.,Proll, S.C., McDermott, J.E., Gritsenko, M.A., Zhang, Q., Zhao, R., Metz, T.O.,Camp II, D.G., Waters, K.M., Smith, R.D., Rice, C.M., Katze, M.G., 2010. Temporalproteome and lipidome profiles reveal hepatitis C virus-associated reprogram-ming of hepatocellular metabolism and bioenergetics. PLoS Pathog. 6,e1000719.

Dolado, I., Swat, A., Ajenjo, N., De Vita, G., Cuadrado, A., Nebreda, A.R., 2007. p38αMAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell11, 191–205.

Elson, D.A., Ryan, H.E., Snow, J.W., Johnson, R., Arbeit, J.M., 2000. Coordinate up-regulation of hypoxia inducible factor (HIF)-1α and HIF-1 target genes duringmulti-stage epidermal carcinogenesis and wound healing. Cancer Res. 60,6189–6195.

Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H.H., Ibrahim-Hashim, A.,Bailey, K., Balagurunathan, Y., Rothberg, J.M., Sloane, B.F., Johnson, J., Gatenby, R.A., Gillies, R.J., 2013. Acidity generated by the tumor microenvironment driveslocal invasion. Cancer Res. 73, 1524–1535.

El-Serag, H.B., 2012. Epidemiology of viral hepatitis and hepatocellular carcinoma.Gastroenterology 142, 1264–1273.

Fallot, G., Neuveut, C., Buendia, M.A., 2012. Diverse roles of hepatitis B virus in livercancer. Curr. Opin. Virol. 2, 467–473.

Fernández-Figueras, M.T., Puig, L., Musulén, E., Gilaberte, M., Lerma, E., Serrano, S.,Ferrándiz, C., Ariza, A., 2006. Expression profiles associated with aggressivebehavior in Merkel cell carcinoma. Mod. Pathol. 20, 90–101.

Feng, H., Shuda, M., Chang, Y., Moore, P.S., 2008. Clonal integration of a poly-omavirus in human Merkel cell carcinoma. Science 319, 1096–1100.

Forsythe, J.A., Jiang, B.H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D., Semenza, G.L.,1996. Activation of vascular endothelial growth factor gene transcription byhypoxia-inducible factor 1. Mol. Cell. Biol. 16, 4604–4613.

Friborg, J., Kong, W., Hottiger, M.O., Nabel, G.J., 1999. p53 inhibition by the LANAprotein of KSHV protects against cell death. Nature 402, 889–894.

Fujiwara, S., Nakagawa, K., Harada, H., Nagato, S., Furukawa, K., Teraoka, M., Seno, T.,Oka, K., Iwata, S., Ohnishi, T., 2007. Silencing hypoxia-inducible factor-1αinhibits cell migration and invasion under hypoxic environment in malignantgliomas. Int. J. Oncol. 30, 793–802.

Garber, K., 2004. Energy boost: the Warburg effect returns in a new theory ofcancer. J. Natl. Cancer Inst. 96, 1805–1806.

Gasperini, P., Espigol-Frigole, G., McCormick, P.J., Salvucci, O., Maric, D., Uldrick, T.S.,Polizzotto, M.N., Yarchoan, R., Tosato, G., 2012. Kaposi sarcoma herpesviruspromotes endothelial-to-mesenchymal transition through Notch-dependentsignaling. Cancer Res. 72, 1157–1169.

Grassmann, R., Aboud, M., Jeang, K.T., 2005. Molecular mechanisms of cellulartransformation by HTLV-1 Tax. Oncogene 24, 5976–5985.

Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell144, 646–674.

Haque, M., Davis, D.A., Wang, V., Widmer, I., Yarchoan, R., 2003. Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) contains hypoxia responseelements: relevance to lytic induction by hypoxia. J. Virol. 77, 6761–6768.

Holotnakova, T., Tylkova, L., Takacova, M., Kopacek, J., Petrik, J., Pastorekova, S.,Pastorek, J., 2010. Role of the HBx oncoprotein in carbonic anhydrase 9 induc-tion. J. Med. Virol. 82, 32–40.

Horikawa, T., Yoshizaki, T., Sheen, T.S., Lee, S.Y., Furukawa, M., 2000. Association oflatent membrane protein 1 and matrix metalloproteinase 9 with metastasis innasopharyngeal carcinoma. Cancer 89, 715–723.

Horikawa, T., Yang, J., Kondo, S., Yoshizaki, T., Joab, I., Furukawa, M., Pagano, J.S.,2007. Twist and epithelial-mesenchymal transition are induced by the EBVoncoprotein latent membrane protein 1 and are associated with metastaticnasopharyngeal carcinoma. Cancer Res. 67, 1970–1978.

Horikawa, T., Yoshizaki, T., Kondo, S., Furukawa, M., Kaizaki, Y., Pagano, J.S., 2011.Epstein–Barr virus latent membrane protein 1 induces snail and epithelial–mesenchymal transition in metastatic nasopharyngeal carcinoma. Br. J. Cancer104, 1160–1167.

Humar, R., Kiefer, F.N., Berns, H., Resink, T.J., Battegay, E.J., 2002. Hypoxia enhancesvascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling. FASEB J. 16, 771–780.

Higgins, D.F., Kimura, K., Bernhardt, W.M., Shrimanker, N., Akai, Y., Hohenstein,B., Saito, Y., Johnson, R.S., Kretzler, M., Cohen, C.D., Eckardt, K.-U., Iwano, M.,Haase, V.H., 2007. Hypoxia promotes fibrogenesis in vivo via HIF-1stimulation of epithelial-to-mesenchymal transition. J. Clin. Invest. 117,3810–3820.

Hui, E.P., Chan, A.T.C., Pezzella, F., Turley, H., To, K.-F., Poon, T.C.W., Zee, B., Mo, F.,Teo, P.M.L., Huang, D.P., Gatter, K.C., Johnson, P.J., Harris, A.L., 2002. Coexpres-sion of hypoxia-inducible factors 1α and 2α, carbonic anhydrase IX, andvascular endothelial growth factor in nasopharyngeal carcinoma and relation-ship to survival. Clin. Cancer Res. 8, 2595–2604.

Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger, R.H., Gassmann,M., Gearhart, J.D., Lawler, A.M., Yu, A.Y., Semenza, G.L., 1998. Cellular anddevelopmental control of O2 homeostasis by hypoxia-inducible factor 1α.Genes Dev. 12, 149–162.

Jeong, S.J., Dasgupta, A., Jung, K.J., Um, J.H., Burke, A., Park, H.U., Brady, J.N., 2008.PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells. Virology 370, 264–272.

Jham, B.C., Ma, T., Hu, J., Chaisuparat, R., Friedman, E.R., Pandolfi, P.P., Schneider, A.,Sodhi, A., Montaner, S., 2011. Amplification of the angiogenic signal through theactivation of the TSC/mTOR/HIF axis by the KSHV vGPCR in Kaposi’s sarcoma.PLoS One 6, e19103.

Jiang, B.H., Jiang, G., Zheng, J.Z., Lu, Z., Hunter, T., Vogt, P.K., 2001. Phosphatidyli-nositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. CellGrowth Differ. 12, 363–369.

Joyce, J.A., Pollard, J.W., 2009. Microenvironmental regulation of metastasis. Nat.Rev. Cancer 9, 239–252.

Kalla, M., Hammerschmidt, W., 2012. Human B cells on their route to latentinfection–early but transient expression of lytic genes of Epstein-Barr virus.EJCB 91, 65–69.

Kato, H., Tamamizu-Kato, S., Shibasaki, F., 2004. Histone deacetylase 7 associateswith hypoxia-inducible factor 1α and increases transcriptional activity. J. Biol.Chem. 279, 41966–41974.

Kim, C.M., Koike, K., Saito, I., Miyamura, T., Jay, G., 1991. HBx gene of hepatitis Bvirus induces liver cancer in transgenic mice. Nature 351, 317–320.

Kim, J.W., Dang, C.V., 2006. Cancer’s molecular sweet tooth and the Warburg effect.Cancer Res. 66, 8927–8930.

Klingelhutz, A.J., Foster, S.C., McDougall, J.K., 1996. Telomerase activation by the E6gene product of human papillomavirus type 16. Nature 380, 79–82.

Kong, X., Lin, Z., Liang, D., Fath, D., Sang, N., Caro, J., 2006. Histone deacetylaseinhibitors induce VHL and ubiquitin-independent proteasomal degradation ofhypoxia-inducible factor 1α. Mol. Cell. Biol. 26, 2019–2028.

Kondo, S., Seo, S.Y., Yoshizaki, T., Wakisaka, N., Furukawa, M., Joab, I., Jang, K.L., Pagano,J.S., 2006. EBV latent membrane protein 1 up-regulates hypoxia-inducible factor1α through Siah1-mediated down-regulation of prolyl hydroxylases 1 and 3 innasopharyngeal epithelial cells. Cancer Res. 66, 9870–9877.

Kondoh, H., Lleonart, M.E., Gil, J., Wang, J., Degan, P., Peters, G., Martinez, D.,Carnero, A., Beach, D., 2005. Glycolytic enzymes can modulate cellular life span.Cancer Res. 65, 177–185.

Krieg, M., Haas, R., Brauch, H., Acker, T., Flamme, I., Plate, K.H., 2000. Up-regulationof hypoxia-inducible factors HIF-1alpha and HIF-2alpha under normoxicconditions in renal carcinoma cells by von Hippel–Lindau tumor suppressorgene loss of function. Oncogene 19, 5435–5443.

Krishna, S.M., James, S., Balaram, P., 2006. Expression of VEGF as prognosticator inprimary nasopharyngeal cancer and its relation to EBV status. Virus Res. 115,85–90.

Krishnamachary, B., Berg-Dixon, S., Kelly, B., Agani, F., Feldser, D., Ferreira, G., Iyer, N.,LaRusch, J., Pak, B., Taghavi, P., Semenza, G.L., 2003. Regulation of colon carcinomacell invasion by hypoxia-inducible factor 1. Cancer Res. 63, 1138–1143.

Kuo, W.H., Chou, F.P., Lu, S.C., Chu, S.C., Hsieh, Y.S., 2000. Significant differences inserum activities of matrix metalloproteinase-2 and-9 between HCV-and HBV-infected patients and carriers. Clin. Chim. Acta 294, 157–168.

S. Cuninghame et al. / Virology 456-457 (2014) 370–383 381

Lando, D., Peet, D.J., Whelan, D.A., Gormon, J.J., Whitelaw, M.L., 2002. Asparaginehydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295,858–861.

Laughner, E., Taghavi, P., Chiles, K., Mahon, P.C., Semenza, G.L., 2001. HER2 (neu)signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis:novel mechanism for HIF-1-mediated vascular endothelial growth factorexpression. Mol. Cell. Biol. 21, 3995–4004.

Le Buanec, H., D’Anna, R., Lachgar, A., Zagury, J.F., Bernard, J., Ittelé, D.D., d’Alessio,P., Hallez, S., Giannouli, C., Burny, A., Bizzini, B., Gallo, R.C., Zagury, D., 1999.HPV-16 E7 but not E6 oncogenic protein triggers both cellular immunosup-pression and angiogenic processes. Biomed. Pharmacother. 53, 424–431.

Lee, S.W., Lee, Y.M., Bae, S.K., Murakami, S., Yun, Y., Kim, K.W., 2000. Humanhepatitis B virus X protein is a possible mediator of hypoxia-induced angiogen-esis in hepatocarcinogenesis. Biochem. Biophys. Res. Commun. 268, 456–461.

Lee, D.F., Kuo, H.P., Chen, C.T., Hsu, J.M., Chou, C.K., Wei, Y., Sun, H.L., Li, L.Y., Ping, B.,Huang, W.C., He, X., Hung, J.Y., Lai, C.C., Ding, Q., Su, J.L., Yang, J.Y., Sahin, A.A.,Hortobagyi, G.N., Tsai, F.J., Tsai, C.H., Hung, M.C., 2007. IKKβ suppression of TSC1links inflammation and tumor angiogenesis via the mTOR pathway. Cell 130,440–455.

Lee, W.Y., Huang, S.C., Hsu, K.F., Tzeng, C.C., Shen, W.L., 2008. Roles for hypoxia-regulated genes during cervical carcinogenesis: somatic evolution during thehypoxia–glycolysis–acidosis sequence. Gynecol. Oncol. 108, 377–384.

Li, G., He, L., Zhang, E., Shi, J., Zhang, Q., Le, A.D., Zhou, K., Tang, X., 2011.Overexpression of human papillomavirus (HPV) type 16 oncoproteins promotesangiogenesis via enhancing HIF-1α and VEGF expression in non-small cell lungcancer cells. Cancer Lett. 311, 160–170.

Lim, J.H., Lee, Y.M., Chun, Y.S., Chen, J., Kim, J.E., Park, J.W., 2010. Sirtuin 1 modulatescellular responses to hypoxia by deacetylating hypoxia-inducible factor 1alpha.Mol. Cell 38, 864–878.

Lichtinghagen, R., Bahr, M.J., Wehmeier, M., Michels, D., Haberkorn, C.I., Arndt, B.,Flemming, P., Manns, M.P., Boeker, K.H.W., 2003. Expression and coordinatedregulation of matrix metalloproteinases in chronic hepatitis C and hepatitis Cvirus-induced liver cirrhosis. Clin. Sci. 105, 373–382.

Lu, Z.H., Wright, J.D., Belt, B., Cardiff, R.D., Arbeit, J.M., 2007. Hypoxia-induciblefactor-1 facilitates cervical cancer progression in human papillomavirus type 16transgenic mice. Am. J. Pathol. 171, 667–681.

Liu, C., Shi, Y., Han, Z., Pan, Y., Liu, N., Han, S., Chen, Y., Lan, M., Qiao, T., Fan, D., 2003.Suppression of the dual-specificity phosphatase MKP-1 enhances HIF-1 trans-activation and increases expression of EPO. Biochem. Biophys. Res. Commun.312, 780–786.

Liu, J., Ahiekpor, A., Li, L., Li, X., Arbuthnot, P., Kew, M., Feitelson, M.A., 2009.Increased expression of ErbB‐2 in liver is associated with hepatitis B� antigenand shorter survival in patients with liver cancer. Int. J. Cancer 125, 1894–1901.

Liu, L.P., Liang, H.F., Chen, X.P., Zhang, W.G., Yang, S.L., Xu, T., Ren, L., 2010. The roleof NF-κB in Hepatitis B virus X protein-mediated upregulation of VEGF andMMPs. Cancer Invest. 28, 443–451.

Lo, K.W., Lo, Y.D., Leung, S.F., Tsang, Y.S., Chan, L.Y., Johnson, P.J., Hjelm, N.M., Lee, J.C.K., Huang, D.P., 1999. Analysis of cell-free Epstein-Barr virus-associated RNA inthe plasma of patients with nasopharyngeal carcinoma. Clin. Chem. 45,1292–1294.

López-Ocejo, O., Viloria-Petit, A., Bequet-Romero, M., Mukhopadhyay, D., Rak, J.,Kerbel, R.S., 2000. Oncogenes and tumor angiogenesis: the HPV-16 E6 onco-protein activates the vascular endothelial growth factor (VEGF) gene promoterin a p53 independent manner. Oncogene 19, 4611–4620.

Lu, H., Forbes, R.A., Verma, A., 2002. Hypoxia-inducible factor 1 activation byaerobic glycolysis implicates the Warburg effect in carcinogenesis. J. Biol. Chem.277, 23111–23115.

Ma, L., Young, J., Prabhala, H., Pan, E., Mestdagh, P., Muth, D., Teruya-Feldstein, J.,Reinhardt, F., Onder, T.T., Valastyan, S., Westermann, F., Speleman, F., Vande-sompele, J., Weinberg, R.A., 2010. miR-9, a MYC/MYCN-activated microRNA,regulates E-cadherin and cancer metastasis. Nat. Cell Biol. 12, 247–256.

Mas, V.R., Maluf, D.G., Archer, K.J., Yanek, K.C., Fisher, R.A., 2007. Angiogenesissoluble factors as hepatocellular carcinoma noninvasive markers for monitor-ing hepatitis C virus cirrhotic patients awaiting liver transplantation. Trans-plantation 84, 1262–1271.

Majumder, P.K., Febbo, P.G., Bikoff, R., Berger, R., Xue, Q., McMahon, L.M., Manola, J.,Brugarolas, J., McDonnell, T.J., Golub, T.R., Loda, M., Lane, H.A., Sellers, W.R., 2004.mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia throughregulation of apoptotic and HIF-1-dependent pathways. Nat. Med. 10, 594–601.

Masood, R., Cai, J., Zheng, T., Smith, D.L., Naidu, Y., Gill, P.S., 1997. Vascularendothelial growth factor/vascular permeability factor is an autocrine growthfactor for AIDS-Kaposi sarcoma. Proc. Natl. Acad. Sci. USA 94, 979–984.

Matthews, K., Leong, C.M., Baxter, L., Inglis, E., Yun, K., Bäckström, B.T., Doorbar, J.,Hibma, M., 2003. Depletion of Langerhans cells in human papillomavirus type16-infected skin is associated with E6-mediated down regulation of E-cadherin.J. Virol. 77, 8378–8385.

McGivern, D.R., Lemon, S.M., 2011. Virus-specific mechanisms of carcinogenesis inhepatitis C virus associated liver cancer. Oncogene 30, 1969–1983.

Mesri, E.A., Cesarman, E., Boshoff, C., 2010. Kaposi’s sarcoma and its associatedherpesvirus. Nat. Rev. Cancer 10, 707–719.

Minet, E., Ernest, I., Michel, G., Roland, I., Remacle, J., Raes, M., Michiels, C., 1999.HIF1A gene transcription is dependent on a core promoter sequence encom-passing activating and inhibiting sequences located upstream from the tran-scription initiation site and cis elements located within the 5’-UTR. Biochem.Biophys. Res. Commun. 261, 534–540.

Montaner, S., Sodhi, A., Pece, S., Mesri, E.A., Gutkind, J.S., 2001. The Kaposi’ssarcoma-associated herpesvirus G protein-coupled receptor promotes endothe-lial cell survival through the activation of Akt/protein kinase B. Cancer Res. 61,2641–2648.

Moon, E.J., Jeong, C.H., Jeong, J.W., Kim, K.R., Yu, D.Y., Murakami, S., Kim, C.W., Kim,K.W., 2004. Hepatitis B virus X protein induces angiogenesis by stabilizinghypoxia-inducible factor-1α. FASEB J. 18, 382–384.

Moore, P.S., Chang, Y., 2010. Why do viruses cause cancer? Highlights of the firstcentury of human tumour virology. Nat. Rev. Cancer 10, 878–889.

Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K.,Matsuura, Y., Kimura, S., Miyamura, T., Koike, K., 1998. The core protein ofhepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat.Med. 4, 1065–1067.

Murono, S., Inoue, H., Tanabe, T., Joab, I., Yoshizaki, T., Furukawa, M., Pagano, J.S.,2001. Induction of cyclooxygenase-2 by Epstein–Barr virus latent membraneprotein 1 is involved in vascular endothelial growth factor production innasopharyngeal carcinoma cells. Proc. Natl. Acad. Sci. USA 98, 6905–6910.

Niu, G., Briggs, J., Deng, J., Ma, Y., Lee, H., Kortylewski, M., Kujawski, M., Kay, H.,Cress, W.D., Jove, R., Yu, H., 2008. Signal transducer and activator oftranscription 3 is required for hypoxia-inducible factor-1α RNA expression inboth tumor cells and tumor-associated myeloid cells. Mol. Cancer Res. 6,1099–1105.

Nakamura, M., Bodily, J.M., Beglin, M., Kyo, S., Inoue, M., Laimins, L.A., 2009.Hypoxia-specific stabilization of HIF-1alpha by human papillomaviruses. Vir-ology 387, 442–448.

Nasimuzzaman, M., Waris, G., Mikolon, D., Stupack, D.G., Siddiqui, A., 2007.Hepatitis C virus stabilizes hypoxia-inducible factor 1α and stimulates thesynthesis of vascular endothelial growth factor. J. Virol. 81, 10249–10257.

Niccoli, S., Abraham, S., Richard, C., Zehbe, I., 2012. The Asian-American E6 variantprotein of human papillomavirus 16 alone is sufficient to promote immortali-zation, transformation, and migration of primary human foreskin keratino-cytes. J. Virol. 86, 12384–12396.

Noch, E., Khalili, K., 2012. Oncogenic viruses and tumor glucose metabolism: likekids in a candy store. Mol. Cancer Ther. 11, 14–23.

Núñez, O., Fernández-Martínez, A., Majano, P.L., Apolinario, A., Gómez-Gonzalo, M.,Benedicto, I., López-Cabrera, M., Boscá, L., Clemente, G., García-Monzón, C.,Martín-Sanz, P., 2004. Increased intrahepatic cyclooxygenase 2, matrix metal-loproteinase 2, and matrix metalloproteinase 9 expression is associated withprogressive liver disease in chronic hepatitis C virus infection: role of viral coreand NS5A proteins. Gut 53, 1665–1672.

Oka, H., Shiozaki, H., Kobayashi, K., Inoue, M., Tahara, H., Kobayashi, T., Takatsuka, Y.,Matsuyoshi, N., Hirano, S., Takeichi, M., Mori, T., 1993. Expression of E-cadherincell adhesion molecules in human breast cancer tissues and its relationship tometastasis. Cancer Res. 53, 1696–1701.

Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., Weinberg, R.A., 2008. Loss ofE-cadherin promotes metastasis via multiple downstream transcriptional path-ways. Cancer Res. 68, 3645–3654.

O’Neil, J.D., Owen, T.J., Wood, V.H., Date, K.L., Valentine, R., Chukwuma, M.B.,Arrand, J.R., Dawson, C.W., Young, L.S., 2008. Epstein–Barr virus-encoded EBNA1modulates the AP-1 transcription factor pathway in nasopharyngeal carcinomacells and enhances angiogenesis in vitro. J. Gen. Virol. 89, 2833–2842.

Parks, S.K., Chiche, J., Pouysségur, J., 2013. Disrupting proton dynamics and energymetabolism for cancer therapy. Nat. Rev. Cancer 13, 611–623.

Peloponese, J.M., Jeang, K.T., 2006. Role for Akt/protein kinase B and activatorprotein-1 in cellular proliferation induced by the human T-cell leukemia virustype 1 tax oncoprotein. J. Biol. Chem. 281, 8927–8938.

Potter, C., Harris, A.L., 2004. Hypoxia inducible carbonic anhydrase IX, marker oftumour hypoxia, survival pathway and therapy target. Cell Cycle 3, 159–162.

Qian, D.Z., Kachhap, S.K., Collis, S.J., Verheul, H.M., Carducci, M.A., Atadja, P., Pili, R.,2006. Class II histone deacetylases are associated with VHL-independentregulation of hypoxia-inducible factor 1α. Cancer Res. 66, 8814–8821.

Qian, L.W., Xie, J., Ye, F., Gao, S.J., 2007. Kaposi’s sarcoma-associated herpesvirusinfection promotes invasion of primary human umbilical vein endothelial cellsby inducing matrix metalloproteinases. J. Virol. 81, 7001–7010.

Qi, M., Elion, E.A., 2005. MAP kinase pathways. J. Cell Sci. 118, 3569–3572.Radkov, S.A., Kellam, P., Boshoff, C., 2000. The latent nuclear antigen of Kaposi

sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway andwith the oncogene Hras transforms primary rat cells. Nat. Med. 6, 1121–1127.

Ramos da Silva, S., Elgui de Oliveira, D., 2011. HIV, EBV and KSHV: viral cooperationin the pathogenesis of human malignancies. Cancer Lett. 305, 175–185.

Ravi, R., Mookerjee, B., Bhujwalla, Z.M., Sutter, C.H., Artemov, D., Zeng, Q., Dillehay, L.E.,Madan, A., Semenza, G.L., Bedi, A., 2000. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1α. Genes Dev. 14, 34–44.

Ray, R.B., Steele, R., Meyer, K., Ray, R., 1997. Transcriptional repression of p53promoter by hepatitis C virus core protein. J. Biol. Chem. 272, 10983–10986.

Richard, D.E., Berra, E., Gothie, E., Roux, D., Pouyssegur, J., 1999. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1α (HIF-1α) andenhance the transcriptional activity of HIF-1. J. Biol. Chem. 274, 32631–32637.

Ripoli, M., D’Aprile, A., Quarato, G., Sarasin-Filipowicz, M., Gouttenoire, J., Scrima, R.,Cela, O., Boffoli, D., Heim, M.H., Moradpour, D., Capitanio, N., Piccoli, C., 2010.Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1α-mediated glycolytic adaptation. J. Virol. 84, 647–660.

Rius, J., Guma, M., Schachtrup, C., Akassoglou, K., Zinkernagel, A.S., Nizet Johnson, R.S., Haddad, G.G., Karin, M., 2008. NF-κB links innate immunity to the hypoxicresponse through transcriptional regulation of HIF-1α. Nature 453, 807–811.

S. Cuninghame et al. / Virology 456-457 (2014) 370–383382

Rivas, C., Thlick, A.E., Parravicini, C., Moore, P.S., Chang, Y., 2001. Kaposi’s sarcoma-associated herpesvirus LANA2 is a B-cell-specific latent viral protein thatinhibits p53. J. Virol. 75, 429–438.

Robey, I.F., Baggett, B.K., Kirkpatrick, N.D., Roe, D.J., Dosescu, J., Sloane, B.F., Hashim, A.I.,Morse, D.L., Raghunand, N., Gatenby, R.A., Gillies, R.J., 2009. Bicarbonate increasestumor pH and inhibits spontaneous metastases. Cancer Res. 69, 2260–2268.

Rofstad, E.K., Mathiesen, B., Kindem, K., Galappathi, K., 2006. Acidic extracellular pHpromotes experimental metastasis of human melanoma cells in athymic nudemice. Cancer Res. 66, 6699–6707.

Russo, J.J., Bohenzky, R.A., Chien, M.C., Chen, J., Yan, M., Maddalena, D., Parry, J.P.,Peruzzi, D., Edelman, I.S., Chang, Y., Moore, P.S., 1996. Nucleotide sequence ofthe Kaposi sarcoma-associated herpesvirus (HHV8). Proc. Natl. Acad. Sci. USA93, 14862–14867.

Salcedo, X., Medina, J., Sanz‐Cameno, P., García‐Buey, L., Martín‐Vilchez, S., Borque,M.J., López-Cabrera, M., Moreno‐Otero, R., 2005. The potential of angiogenesissoluble markers in chronic hepatitis C. Hepatology 42, 696–701.

Scheffner, M., Huibregtse, J.M., Vierstra, R.D., Howley, P.M., 1993. The HPV-16 E6and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitinationof p53. Cell 75, 495–505.

Schultz, B.E., Chan, S.I., 2001. Structures and proton-pumping strategies of mito-chondrial respiratory enzymes. Annu. Rev. Biophys. Biomol. Struct. 30, 23–65.

Schipper, J.H., Frixen, U.H., Behrens, J., Unger, A., Jahnke, K., Birchmeier, W., 1991.E-cadherin expression in squamous cell carcinomas of head and neck: inversecorrelation with tumor dedifferentiation and lymph node metastasis. CancerRes. 51, 6328–6337.

Semenza, G.L., Roth, P.H., Fang, H.M., Wang, G.L., 1994. Transcriptional regulation ofgenes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem.269, 23757–23763.

Semenza, G.L., 2000. HIF-1: mediator of physiological and pathophysiologicalresponses to hypoxia. J. Appl. Physiol. 88, 1474–1480.

Semenza, G.L., 2001. HIF-1, O2, and the 3 PHDs: how animal cells signal hypoxia tothe nucleus. Cell 107, 1–3.

Semenza, G.L., 2003. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732.Semenza, G.L., 2007. HIF-1 mediates the Warburg effect in clear cell renal

carcinoma. J. Bioenerg. Biomembr. 39, 231–234.Semenza, G.L., 2010. HIF-1: upstream and downstream of cancer metabolism. Curr.

Opin. Genet. Dev. 20, 51–56.Semenza, G.L., 2012. Hypoxia-inducible factors: mediators of cancer progression

and targets for cancer therapy. Trends Pharmacol. Sci. 33, 207–214.Shaw, R.J., 2006. Glucose metabolism and cancer. Curr. Opin. Cell Biol. 18, 598–608.Shin, Y.C., Joo, C.H., Gack, M.U., Lee, H.R., Jung, J.U., 2008. Kaposi’s sarcoma-

associated herpesvirus viral IFN regulatory factor 3 stabilizes hypoxia-inducible factor-1 alpha to induce vascular endothelial growth factor expres-sion. Cancer Res. 68, 1751–1759.

Sodhi, A., Montaner, S., Patel, V., Zohar, M., Bais, C., Mesri, E.A., Gutkind, J.S., 2000.The Kaposi’s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion throughmitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1α. Cancer Res. 60, 4873–4880.

Son, Y., Cheong, Y.K., Kim, N.H., Chung, H.T., Kang, D.G., Pae, H.O., 2011. Mitogen-activated protein kinases and reactive oxygen species: how can ROS activateMAPK pathways? J. Signal Transduct. 2011, 6.

Sperandio, S., Fortin, J., Sasik, R., Robitaille, L., Corbeil, J., de Belle, I., 2009. Thetranscription factor Egr1 regulates the HIF‐1α gene during hypoxia. Mol.Carcinogen. 48, 38–44.

Steeg, P.S., 2006. Tumor metastasis: mechanistic insights and clinical challenges.Nat. Med. 12, 895–904.

Stroka, D.M., Burkhardt, T., Desbaillets, I., Wenger, R.H., Neil, D.A.H., Bauer, C.,Gassmann, M., Candinas, D., 2001. FASEB J. 15, 2445–2453.

Suzuki, H., Tomida, A., Tsuruo, T., 2001. Dephosphorylated hypoxia-inducible factor1alphaas a mediator of p53-dependent apoptosis during hypoxia. Oncogene 20,5779–5788.

Swinson, D.E., Jones, J.L., Richardson, D., Wykoff, C., Turley, H., Pastorek, J., Taub, N.,Harris, A.L., O’Byrne, K.J., 2003. Carbonic anhydrase IX expression, a novelsurrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer. J. Clin. Oncol. 21, 473–482.

Švastová, E., Hulıková, A., Rafajová, M., Zat’ovičová, M., Gibadulinová, A., Casini, A.,Cecchi, A., Scozzafava, A., Supuran, C.T., Pastorek, J., Pastoreková, S., 2004.Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX toacidify extracellular pH. FEBS Lett. 577, 439–445.

Tardif, K.D., Waris, G., Siddiqui, A., 2005. Hepatitis C virus, ER stress, and oxidativestress. Trends Microbiol. 13, 159–163.

Thompson, M.P., Kurzrock, R., 2004. Epstein–Barr virus and cancer. Clin. Cancer Res.10, 803–821.

Tang, X., Zhang, Q., Nishitani, J., Brown, J., Shi, S., Le, A.D., 2007. Overexpression ofhuman papillomavirus type 16 oncoproteins enhances hypoxia-inducible factor1α protein accumulation and vascular endothelial growth factor expression inhuman cervical carcinoma cells. Clin. Cancer Res. 13, 2568–2576.

Thomas, G.V., Tran, C., Mellinghoff, I.K., Welsbie, D.S., Chan, E., Fueger, B., Czernin, J.,Sawyers, C.L., 2005. Hypoxia-inducible factor determines sensitivity to inhibi-tors of mTOR in kidney cancer. Nat. Med. 12, 122–127.

Tomita, M., Semenza, G., Michiels, C., Matsuda, T., Uchihara, J., Okudaira, T., Tanaka,Y., Taira, N., Ohshiro, K., Mori, N., 2007. Activation of hypoxia-inducible factor1 in human T-cell leukaemia virus type 1-infected cell lines and primary adultT-cell leukaemia cells. Biochem. J. 406, 317–323.

Turk, V., Turk, B., Turk, D., 2001. Lysosomal cysteine proteases: facts and opportu-nities. EMBO J. 20, 4629–4633.

Umbas, R., Isaacs, W.B., Bringuier, P.P., Schaafsma, H.E., Karthaus, H.F., Oosterhof, G.O., Debruyne, F.M., Schalken, J.A., 1994. Decreased E-cadherin expression isassociated with poor prognosis in patients with prostate cancer. Cancer Res. 54,3929–3933.

van Uden, P., Kenneth, N.S., Rocha, S., 2008. Regulation of hypoxia-inducible factor-1α by NF-κB. Biochem. J. 412, 477–484.

Vander Heiden, M.G., Cantley, L.C., Thompson, C.B., 2009. Understanding theWarburg effect: the metabolic requirements of cell proliferation. Science 324,1029–1033.

Wakisaka, N., Pagano, J.S., 2003. Epstein–Barr virus induces invasion and metastasisfactors. Anticancer Res. 23, 2133–2138.

Wakisaka, N., Kondo, S., Yoshizaki, T., Murono, S., Furukawa, M., Pagano, J.S., 2004.Epstein–Barr virus latent membrane protein 1 induces synthesis of hypoxia-inducible factor 1α. Mol. Cell. Biol. 24, 5223–5234.

Wang, G.L., Jiang, B.H., Rue, E.A., Semenza, G.L., 1995a. Hypoxia-inducible factor 1 isa basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc. Natl. Acad. Sci. USA 92, 5510–5514.

Wang, X., Martindale, J., Liu, Y., Holbrook, N., 1998. The cellular response tooxidative stress: influences of mitogen-activated protein kinase signallingpathways on cell survival. Biochem. J. 333, 291–300.

Wang, L., Wakisaka, N., Tomlinson, C.C., DeWire, S.M., Krall, S., Pagano, J.S.,Damania, B., 2004. The Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) K1 protein induces expression of angiogenic and invasion factors. Cancer Res.64, 2774–2781.

Warburg, O., Wind, F., Negelein, E., 1927. The metabolism of tumors in the body. J.Gen. Physiol. 8, 519–530.

Wen, K.W., Damania, B., 2010. Kaposi sarcoma-associated herpesvirus (KSHV):molecular biology and oncogenesis. Cancer Lett. 289, 140–150.

Wenger, R.H., Rolfs, A., Marti, H.H., Guenet, J.-L., Gassmann, M., 1996. Nucleotidesequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor-1 α. Biochem. Biophys. Res. Commun. 223, 54–59.

Wenger, R.H., Kvietikova, I., Rolfs, A., Gassmann, M., Marti, H.H., 1997. Hypoxia-inducible factor-1α is regulated at the post mRNA level. Kidney Int. 51, 560–563.

Wiener, C.M., Booth, G., Semenza, G.L., 1996. In vivo expression of mRNAs encodinghypoxia-inducible factor 1. Biochem. Biophys. Res. Commun. 225, 485–488.

Wilson, G.K., Brimacombe, C.L., Rowe, I.A., Reynolds, G.M., Fletcher, N.F., Stamataki,Z., Bhoagi, R.H., Simões, M.L., Ashcroft, M., Afford, S.C., Mitry, R.R., Dhawan, A.,Mee, C.J., Hübscher, S.G., Bafle, P., McKeating, J.A., 2012. A dual role for hypoxiainducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration.J. Hepatol. 56, 803–809.

Wise-Draper, T.M., Wells, S.I., 2007. Papillomavirus E6 and E7 proteins and theircellular targets. Front. Biosci. 13, 1003–1017.

Yang, M.H., Wu, K.J., 2008. TWIST activation by hypoxia inducible factor-1 (HIF-1):implications in metastasis and development. Cell Cycle 7, 2090–2096.

Yang, M.H., Chen, C.L., Chau, G.Y., Chiou, S.H., Su, C.W., Chou, T.Y., Peng, W.L., Wu, J.C., 2009. Comprehensive analysis of the independent effect of twist and snail inpromoting metastasis of hepatocellular carcinoma. Hepatology 50, 1464–1474.

Yen, C.J., Lin, Y.J., Yen, C.S., Tsai, H.W., Tsai, T.F., Chang, K.Y., Chang, T.T., 2012. Hepatitis Bvirus X protein upregulates mTOR signaling through IKKβ to increase cellproliferation and VEGF production in hepatocellular carcinoma. PLoS One 7, e41931.

Yoo, Y.G., Oh, S.H., Park, E.S., Cho, H., Lee, N., Park, H., Kim, D.K., Yu, D.Y., Seong, J.K.,Lee, M.O., 2003. Hepatitis B virus X protein enhances transcriptional activity ofhypoxia-inducible factor-1α through activation of mitogen-activated proteinkinase pathway. J. Biol. Chem. 278, 39076–39084.

Yoo, Y.G., Cho, S., Park, S., Lee, M.O., 2004. The carboxy-terminus of the hepatitis Bvirus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1α. FEBS Lett. 577, 121–126.

Yoo, Y.G., Kong, G., Lee, M.O., 2006. Metastasis-associated protein 1 enhancesstability of hypoxia-inducible factor-1alpha protein by recruiting histonedeacetylase 1. EMBO J. 25, 1231–1241.

Yoo, Y.G., Na, T.Y., Seo, H.W., Seong, J.K., Park, C.K., Shin, Y.K., Lee, M.O., 2008.Hepatitis B virus X protein induces the expression of MTA1 and HDAC1, whichenhances hypoxia signaling in hepatocellular carcinoma cells. Oncogene 27,3405–3413.

Yoshizaki, T., Sato, H., Furukawa, M., Pagano, J.S., 1998. The expression of matrixmetalloproteinase 9 is enhanced by Epstein–Barr virus latent membraneprotein 1. Proc. Natl. Acad. Sci. USA 95, 3621–3626.

Young, L.S., Rickinson, A.B., 2004. Epstein–Barr virus: 40 years on. Nat. Rev. Cancer4, 757–768.

Zehbe, I., Wilander, E., Delius, H., Tommasino, M., 1998. Human papillomavirus 16E6 variants are more prevalent in invasive cervical carcinoma than theprototype. Cancer Res. 58, 829–833.

Zhong, H., De Marzo, A.M., Laughner, E., Lim, M., Hilton, D.A., Zagzag, D., Buechler,P., Isaacs, W.B., Semenza, G.L., Simons, J.W., 1999. Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases. CancerRes. 59, 5830–5835.

zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, G., Henle, W., Clifford, P.,Santesson, L., 1970. Epstein–Barr virus in Burkitt’s lymphoma and nasopharyngealcarcinoma. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas ofthe nasopharynx. Nature 228, 1056–1058.

zur Hausen, H., 2002. Papillomaviruses and cancer: from basic studies to clinicalapplication. Nat. Rev. Cancer 2, 342–350.

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