i

ii

iii

Acknowledgments

I would like to thank my supervisor, Michael Parkhouse, not only for all the support and advice given throughout all these years, but also for making us laugh, for the cookies and chocolates and world music to balance my heavy metal tendencies. In summary, thank you for the unique and special working environment.

A special thanks to Rute Nascimento, my “supervisor in the bench”, for everything that she taught me and for the contagious enthusiasm with science. I am very lucky indeed, for the opportunity to work with you.

To everyone from Infection and Immunity group, past and present, specially to Silvia Correia and Sónia Ventura, for sharing with me the good days and the bad years in science.

I would like to acknowledge Instituto Gulbenkian de Ciência, one of the best places not only for post-docs but also for PhD students.

To my friend, Andreia, because after four thesis, and everything in between, the desert is still the same.

I would like to thank all my friends from “Cacém e arredores” not only for their friendship, but also for letting me know how non-scientists live and think, and especially to Pedro Ferreira and Silvia Batista for forming an efficient moral support group. From different research areas, but with the same PhD problem (and some others..) to solve.

À minha familia, especialmente os meus pais, por continuarem a apoiar a minha escolha de “profissão”. Sem eles, não teria conseguido chegar a esta fase, e por isso, o meu muito obrigado!

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Abbreviations:

BAC bacterial artificial chromosome CDK cyclin-dependent kinases CLT cytomegalovirus latency-expressed transcripts CPE cytopathogenic effect DAI DNA-dependent activator of IFN ‑regulatory factors EBV Epstein-Barr virus GM-P granulocyte-monocyte precursor cells GPCR seven-transmembrane G protein-coupled receptor HA Haemaglutinin peptide HCMV Human cytomegalovirus HFF Human foreskin fibroblast HSV-1 Herpes simplex virus 1 HSV-2 Herpes simplex virus 2 IE Immediate early IFN interferon IL-8 Interleukin-8 KSHV Kaposi’s sarcoma-associated herpesvirus LAT latency-associated transcripts MCMV Murine cytomegalovirus MHC major histocompatibility complex MHV-68 Murine herpesvirus strain 68 MIEP major immediate-early promoter region MOI Multiplicity of infection NEMO NF-κB essential modulator NHEJ non-homologous end-joining NK Natural killer PAMP pathogen-associated molecular patterns PBS Phosphate buffered saline PRR pattern-recognition receptors PVDF Polyvinylidene difluoride SDS-PAGE sodium dodecyl sulfate polyacrylamide gel TLR Toll ‑like receptors TNF-α tumor necrosis factor alpha VZV Varicella-zoster virus

v

Resumo

O Citomegalovírus Humano (HCMV), incluído na subfamília dos β- Herpesvírus, infecta indivíduos saudáveis, geralmente, de forma assintomática, no entanto, em indivíduos imunossuprimidos, como pacientes com SIDA, pode provocar doenças graves ou fatais. Após uma infecção primária, o HCMV estabelece uma infecção latente com reactivações periódicas. De modo a assegurar a sua sobrevivência e propagação, o HCMV desenvolveu vários mecanismos para subverter a resposta da imunidade inata e adaptativa do hospedeiro. Infecção pelo HCMV induz a produção da interleucina-8 (IL-8), uma quimiocina pro-inflamatória quimiotáctica, principalmente, para neutrófilos. Os neutrófilos têm um papel importante na disseminação do HCMV ao transportarem o vírus para diferentes órgãos do corpo e transmitindo-o a outros tipos de células. Por outro lado, a IL-8 aumenta a replicação e produção de viriões do HCMV.

Neste estudo, identificou-se o gene UL76 do HCMV como indutor da expressão de IL-8, ao nível da transcrição e da produção de proteína. Este trabalho teve como objectivo principal a caracterização do mecanismo de indução de IL-8 pelo gene UL76 e o seu impacto durante a infecção viral. A activação da transcrição de IL-8 foi abolida na presença de uma mutação no local de ligação ao factor de transcrição NF-kB no promotor da IL-8 do repórter usado em ensaios de luciferase, indicando que a activação de NF-kB é essencial para a indução de IL-8 pelo gene UL76. Do mesmo modo, foi demonstrado que esta indução requer a actividade catalítica de IKK-β e a degradação de Ik βα . Consistente com estes resultados, a expressão do gene UL76 promoveu a translocação da subunidade do NF-kB, p65, para o núcleo. O facto de anteriormente ter sido demonstrado

vi que o gene UL76 induz danos no DNA e paragem do ciclo celular é particularmente relevante para este trabalho, tendo em conta que a via de sinalização do NF-kB pode ser activada pelo reconhecimento de danos no DNA. De facto, não se observou indução de IL-8 pelo gene UL76 na ausência de ATM (células com mutação no gene ATM) ou após inibição da proteína ATM (inibidor específico para ATM, KU55933). De maior importância clínica, é o facto de infecção de células ATM -/- com o HCMV induzir níveis de IL-8 reduzidos comparando com a infecção de células normais. Por outro lado, um vírus HCMV com uma mutação no gene UL76 é significativamente menos eficiente a induzir a expressão de IL-8.

Em conclusão, neste trabalho é identificada uma nova função do gene UL76, indução da expressão de IL-8 através do reconhecimento de danos no DNA e consequente activação da via do NF-kB, e demonstrou-se o papel importante que o gene UL76 e a proteína ATM apresentam na indução de IL-8 durante a infecção pelo HCMV.

vii

Summary:

Human cytomegalovirus (HCMV) is a β-herpesvirus that infects healthy individuals, usually asymptomatically, but can cause severe or fatal disease in immunocompromised individuals such as transplant recipients or AIDS patients. Primary HCMV infection, as with other herpesviruses, is followed by establishment of lifelong latency and periodic reactivation. To ensure its survival and propagation within the host, HCMV has evolved many strategies to subvert both innate and adaptive host immunity. It is known that HCMV infection induces production of interleukin-8 (IL-8), a pro- inflammatory chemokine with neutrophil chemotatic activity. Significantly, neutrophils are a major carrier of HCMV during viremia and they are able to transmit infectious virus to other cells, playing a key role in virus dissemination through their contact with endothelial cells. In addition, IL-8 enhances HCMV virion production. This work has identified an HCMV gene (UL76), with the relevant property of inducing IL-8 expression at both transcriptional and protein levels. Interestingly, the murine homologue, MHV-68 ORF20, has no significant effect in the expression of IL-8. The main objective of this work was to characterize the mechanism of IL-8 induction by UL76 and the impact of its expression during the viral infection. The UL76-mediated enhancement of luciferase activity was abolished when the NF-kB binding element was mutated in the IL-8 promoter luciferase reporter, thereby demonstrating that activation of NF-kB is essential for the UL76-mediated induction of IL-8. Consistent with the requirement for NF-kB pathway activation, IKK-β and degradation of Ik βα were essential for IL-8 induction by UL76. In addition, and as might be predicted, expression of UL76 resulted in the translocation of p65 to the nucleus. Of particular relevance, we and Wang’s group

viii demonstrated that UL76 also induces cell cycle arrest and DNA damage. Here, and as expected from the activation of the NF-kB pathway by the DNA damage response, we show the requirement of ATM in IL-8 induction by UL76. Specifically, there was no induction of IL-8 by UL76 using an ATM -/- cell line and a specific ATM inhibitor, KU55933. More importantly, there was a significant reduction of IL-8 secretion when ATM -/- cells were infected with wild type HCMV virus, suggesting that ATM is involved in IL-8 induction by the virus. To further demonstrate the impact of UL76 on HCMV-induced IL-8, we have established that a UL76 deletion mutant HCMV was significantly less efficient in stimulating IL-8 production than the wild type virus. In conclusion, we demonstrate that expression of UL76 gene induces IL-8 expression as a result of DNA damage and that both UL76 and ATM have a role in the mechanism of IL-8 induction during HCMV infection.

ix

Acknowledgments i Abbreviations ii Resumo iii Summary v Table of contents

Chapter 1: Intro duction 1.1. Virus 1 1.1.1. Herpesviruses 1 1.1.1.1. Virus structure and genome organization 1 1.1.1.2. Life cycle 2 1.1.1.3. Classification 3 1.1.1.3.1. The Alphaherpesvirinae subfamily 5 1.1.1.3.2. The Gammaherpesvirinae subfamily 6 1.1.1.3.3. The Betaherpesvirinae subfamily 6 1.1.1.3.3.1. Human Cytomegalovirus (HCMV) 7 1.1.1.4. The UL24 gene family 10 1.2. HCMV-host interaction 14 1.2.1. Virus modulation of cell cycle and DNA damage 14 1.2.2. Host immune response to virus infection 20 1.2.3. Virus modulation of immune system 26 1.2.4. Virus modulation of chemokines 29 1.2.4.1. IL-8 30 1.2.4.1.1. Gene regulation 31 1.2.4.1.1.1. NF-KB 32 1.2.4.1.1.2. NF-kB Canonical pathway 34 1.2.4.1.1.3. NF-kB Non-Canonical pathway 34 1.2.4.1.1.4. NF-kB activation by genotoxic stress 35 1.2.4.1.2. IL-8 and HCMV 37

x

1.3 Aim of the project 39 1.4. References 40

Chapter 2: Human Cytomegalovirus UL76 protein induces

IL-8 expression 2.1. Summary 56 2.2. Introduction 57 2.3. Materials and Methods 59 2.3.1. Cells 59 2.3.2. Plasmids 59 2.3.3. Lentivirus production and titration 60 2.3.4. Lentivirus transduction and RNA isolation 60 2.3.5. Target Synthesis and Hybridization to Affymetrix 61 GeneChips 2.3.6. Microarrays Data Analysis 62 2.3.7. Reverse transcriptase polymerase chain reaction 62 (RT-PCR) 2.3.8. Luciferase assays 63 2.3.9. Enzyme-linked Immunoabsorbent Assay (ELISA) 63 2.3.10. Western blot 64 2.3.11. Statistical Analysis 64 2.4. Results 64 2.4.1. The HCMV UL76 gene induces transcription of IL-8 64 2.4.2. HCMV UL76 gene induces secretion of IL-8 protein 67 2.5. Discussion 68 2.6. References 70

xi

Chapter 3: Mechanism of IL -8 induction by Human

Cytomegalovirus UL76 protein 3.1. Summary 73 3.2. Introduction 74 3.3. Materials and Methods 76 3.3.1. Cells 76 3.3.2. Plasmids 76 3.3.3. Luciferase assays 77 3.3.4. Immunofluorescence 77 3.3.5. Nuclear extracts preparation 78 3.3.6. Western blot 78 3.3.7. Enzyme-Linked Immunoabsorbent Assay (ELISA) 79 3.3.8. Cell cycle analysis 79 3.3.9. Statistical analysis 80 3.4. Results 80 3.4.1. Induction of IL-8 by HCMV UL76 is NF-kB- 80 dependent 3.4.2. Induction of IL-8 by UL76 requires IKK β activation 82 and IkB α degradation. 3.4.3. UL76 induces translocation of p65 to the nucleus 83 3.4.4. IL-8 induction by HCMV UL76 is ATM-dependent 86 3.4.5. UL76 endonuclease motifs mutation reduced IL-8 90 induction and has no effect on cell cycle arrest 3.5. Discussion 92 3.6. Acknowledgements 95 3.7. References 95

xii

Chapter 4: Mechanism of IL -8 induction by Human

Cytomegalovirus and impact of the UL76 gene 4.1. Summary 99 4.2. Introduction 100 4.3. Materials and Methods 101 4.3.1. Cells 101 4.3.2. HCMV stock production 101 4.3.3. Enzyme-linked Immunoabsorbent Assay (ELISA) 102 4.3.4. Western blot 103 4.4. Results 103 4.4.1. Induction of IL-8 by HCMV is reduced in the 103 absence of UL76 4.4.2. ATM is required for IL-8 induction by HCMV 105

4.5. Discussion 106 4.6. References 109

Chapter 5: Final Considerations 112

Annex. Microarray analysis

xiii

Index of Figures and Tables

Figure 1.1. Canonical and non-canonical signaling to NF-κB 33

Figure 1.2. Activation of NF-κB pathway by genotoxic stress 36

Figure 2.1. UL76 induces IL-8 gene transcription. 65

Figure 2.2. UL76 activates IL-8 promoter transcription. 66

Figure 2.3. UL76 induces IL-8 secretion. 67

Figure 3.1 IL-8 induction by HCMV UL76 is NF-kB-dependent. 81

Figure 3.2 Induction of IL-8 by UL76 requires IKK β activation 83 and IkB α degradation.

Figure 3.3 UL76 induces p65 translocation to the nucleus. 85

Figure 3.4 UL76 activates NEMO through DNA damage 87 pathway.

Figure 3.5 IL-8 induction by UL76 is ATM-dependent. 89

Figure 3.6. Putative endonuclease activity impact on UL76 92 functions.

Figure 4.1 Requirement of UL76 for optimal IL-8 induction by 104 HCMV.

Figure 4.2 IL-8 induction by HCMV is ATM-dependent. 106

Table 1. Human herpesviruses 5

Table 2. The human herpesvirus UL24 gene family 10

xiv

CHAPTER 1

INTRODUCTION

Chapter 1

1.1. Virus

Viruses are obligate intracellular parasites that present uniquely close relationships with the organisms that they infect. Viruses have a limited coding genome thus, they rely on the infected cell to supply the energy, chemicals and much of the machinery required for their replication. Their success, like all parasites, is dependent on the balance that they maintain with their hosts, with which they need to coexist. The different components involved in this equilibrium will be reviewed in this introduction, with specific focus on herpesviruses, a classical example of a successful group of viruses.

1.1.1. Herpesviruses

Herpesviruses are a large group of successful, and widely distributed, double stranded DNA viruses of serious medical and veterinary importance. Although herpesviruses are well adapted to their natural host, their infection can result in severe diseases, particularly in children and immunocompromised individuals (Pellett & Roizman, 2007). Herpesviruses are characterized by their distinct virion morphology and the ability to establish latency during their life cycle.

1.1.1.1. Virus structure and genome organization

Morphologically distinct from all other viruses, a typical herpes virion is spherical and includes four components: the core, the capsid, the tegument and the envelope. The core contains the linear double-stranded DNA genome within an icosahedral protein capsid. Surrounding the capsid is the tegument, a protein matrix,

1

Chapter 1 enclosed by the lipid envelope containing several glycoproteins (Davison, 2007b).

The variability of genome organization of herpesviruses provides the basis for their classification into six groups, ranging from simple forms showing a large sequence repeated at both terminus (e.g. HHV-6), or without repetitions at all (e.g. tupaia herpesvirus), to more complex organizations where the terminus sequences are internally repeated in an inverted orientation, dividing the genomes into two segments, each one consisting in unique sequences flanked by inverted repeats (e.g. HSV-1 and HCMV) (Pellett & Roizman, 2007).

1.1.1.2. Life cycle A common biological property of herpesviruses is their biphasic infection cycle consisting of replicative (lytic) and latent phases. During the lytic phase and viral reactivation, most of viral genes are expressed in a cascade manner, beginning with the immediate- early (IE) genes that do not require viral protein synthesis for their expression. The early (E) genes, whose expression occurs in the presence of IE proteins and before DNA replication, include mainly enzymes involved in nucleotide metabolism and DNA replication. In contrast, expression of late (L) genes is completely dependent on viral DNA synthesis and encodes mainly virion proteins. Additionally, some genes are expressed with leaky-late kinetics and their expression, although augmented by the onset of viral DNA synthesis, is not totally DNA dependent (Davison, 2007a; b). In contrast to other viruses, herpesviruses encode a large array of enzymes involved in nucleic acid metabolism (e.g. thymidine kinase), DNA synthesis (e.g. DNA polymerase), and postsynthetic

2

Chapter 1 modifications of proteins (e.g. protein kinases), although it can vary between different herpesviruses (Pellett & Roizman, 2007). Viral replication occurs in the nucleus by circularization, followed by production of concatemers and cleavage of unit-length molecules which are packaged into the capsids (Boehmer & Lehman, 1997). The assembly of the capsid and packaging of the replicated viral genome occurs in the nucleus. The nucleocapsid is translocated to the cytoplasm by budding at the inner nuclear membrane followed by fusion of the primary envelope with the outer nuclear membrane. This process is mediated by viral and cellular proteins and involves reorganization of the nuclear architecture. During the final maturation process in the cytoplasm, tegument proteins associate with the translocated nucleocapsid and with the future envelope containing viral membrane proteins. This complex network of interactions results in the formation of an infectious virion, released by infected cells during the lytic phase of herpesviruses infection (Mettenleiter et al. , 2009) . Latency, on the other hand, is characterized by limited gene expression and lack of virion production. During this phase, viral genomes form closed circular molecules that retain the capacity to replicate upon reactivation. Although all herpesviruses known are able to establish a latent infection in a specific type of cells, the mechanisms for establishment, maintenance, or termination of the latent phase differ between the viruses (Pellett & Roizman, 2007).

1.1.1.3. Classification

A recent taxonomic classification of herpesviruses defined three families within the order Herpesvirales : Herpesviridae , divided into the alpha, beta, and gamma herpesvirinae subfamilies and

3

Chapter 1 containing herpesviruses that infect mammalian, reptilian and avian hosts; Alloherpesviridae , which infect fish and amphibian hosts; and Malacoherpesviridae , with only one member, targeting the invertebrate bivalve mollusk. Eight different herpesviruses, encompassing all three herpesvirinae subfamilies, are known to infect humans (Davison et al. , 2009). Based on phylogenetic studies the estimated date for the divergence from the most recent common ancestor of the three Herpesvirinae subfamilies is around 400 million years ago (McGeoch & Gatherer, 2005). Another study, based on the number of shared functions between the different subfamilies, supported previous observations of an early split, around 220 million years ago, of the β- and γ-herpesviruses from the α-herpesviruses (Albà et al. , 2001).

The three subfamilies of Herpesviridae are defined according to their different biological proprieties such as host range and growth kinetics. The herpesviruses core genes consist of approximately 40 genes which are conserved in the alpha-, beta-, and gamma- herpesviruses and are mostly involved in fundamental aspects of viral lytic replication (Davison et al. , 2002). Although members of the three subfamilies share a common life style initiated with an acute infection, the process of latency establishment and the cell type where it occurs differs drastically between subfamilies (Table 1). Thus it is not surprising that a large number of genes are conserved only at the subfamily level. The varied pathology of these different groups of herpesviruses is typically associated with reactivation of a persistent infection and the subsequent production of virus (Pellett & Roizman, 2007).

4

Chapter 1

Subfamily Virus Disease (ex.) Latency

Human Herpesvirus 1 (HSV-1) Fever, blisters Sensory ganglia alpha Human Herpesvirus 2 (HSV-2) Genital ulceration neurons

Human Herpesvirus 3 (VZV) Chicken pox, shingles

Human Herpesvirus 5 (HCMV) Mononucleosis beta Human Herpesvirus 6 Mononuclear cells Skin rash Human Herpesvirus 7

Human Herpesvirus 4 (EBV) Burkitt’s lymphoma gamma Lymphocytes Human Herpesvirus 8 (KHSV) Kaposi’s sarcoma

Table 1. Human herpesviruses (Pellett & Roizman, 2007).

A brief description of each subfamily will be presented, focusing on the human herpesviruses and, with emphasis, on human cytomegalovirus (HCMV).

1.1.1.3.1. The Alphaherpesvirinae subfamily

This subfamily contains the genera Simplexvirus (HSV-1 and HSV- 2) and Varicellovirus (VZV) which infect mammalian hosts and, in addition, other genera that infect avian hosts (Davison et al. , 2009).

The α-herpesviruses are characterized by their relatively short life cycle and variable host range. Alpha-herpesviruses latency is established in sensory ganglia and neurons. During this phase, specific latency-associated transcripts (LATs) are expressed and there is no production of infectious virus. In the case of VZV, however, there is only expression of a restricted number of

5

Chapter 1 transcripts and proteins that are present in early phases of normal lytic infection (Hay & Ruyechan, 2007).

1.1.1.3.2. The Gammaherpesvirinae subfamily

The human γ-herpesviruses are divided in two genera, Lymphocryptovirus which includes human Epstein–Barr virus (EBV) and Rhadinovirus , which includes human Kaposi’s sarcoma- associated herpesvirus (KSHV). Recently, two lineages that are separable from the established genera were formed: Macavirus and Percavirus (Davison et al. , 2009) .

The members of this subfamily have cellular tropism for lymphocytes and are characterized by their ability to maintain latent infection in quiescent and proliferating cells. In addition, γ- herpesviruses are able to induce lymphoproliferation and cancers. Tumors associated with EBV and KSHV include mainly lymphoproliferative diseases and lymphomas (Pellett & Roizman, 2007).

1.1.1.3.3. The Betaherpesvirinae subfamily

The β-herpesviruses are divided in two major lineages which include the cytomegaloviruses ( Cytomegalovirus and Muromegalovirus genera), typified by human cytomegalovirus (HCMV), and the Roseolovirus genus (HHV-6 and HHV-7). The recently added genus Proboscivirus recognizes a distinct lineage of the subfamily containing the new species Elephantid herpesvirus 1 (Davison et al. , 2009).

6

Chapter 1

The replication cycle of β-herpesviruses is relatively long, with a slow progression in culture, in contrast to α-herpesviruses. All members of the subfamily have a restricted host range and establish latency in hematopoietic cells in the myeloid lineage (Mocarski et al. , 2007; Sissons et al. , 2002). Both variants of HHV- 6 and HHV-7 are predominately T-lymphotropic, although HHV-6 can also infect cells of myeloid lineage (Lusso et al. , 1994; Santoro et al. , 1999).

1.1.1.3.3.1. Human Cytomegalovirus (HCMV)

Human cytomegalovirus is the largest herpesvirus known, with a genome of approximately 235 kbp encoding more than 200 genes (Mocarski et al. , 2007). The importance of HCMV as a human pathogen is emphasized by its prevalence in the population (estimated to be 50-90% in developed countries), and the fact that HCMV is the leading cause of congenital viral infection in humans. Primary infection with HCMV is frequently asymptomatic in immunocompetent individuals. In immunocompromised patients, however, such as transplant recipients or AIDS patients, HCMV infections frequently result in severe, even fatal, disease (Sissons & Carmichael, 2002). Cells infected with HCMV show a unique cytopathogenic effect (CPE) consisting in rounded and enlarged cells, a characteristic designated cytomegaly which is responsible for the virus name (Albrecht & Weller, 1980).

Infection with HCMV is restricted to humans, but involves a broad range of target cells with epithelial cells, endothelial cells,

7

Chapter 1 fibroblasts and smooth muscle cells being the major targets for virus replication. Productive replication in such ubiquitous cell types permits HCMV to replicate in nearly every organ in the human host (Sinzger et al. , 2008). Fibroblasts are the standard cell culture system for propagation of HCMV, but several strains have endothelial cell tropism. It has been observed that extended propagation of HCMV in fibroblasts frequently results in loss of endothelial cell tropism, whereas propagation in endothelial cells maintains the broad cell tropism of the respective strain (Waldman et al. , 1991). Differences between the cell tropisms of HCMV strains from clinical isolates from different patients may be related to the variable clinical profile of HCMV infections (Sinzger et al. , 1999).

A common characteristic of herpesviruses is their ability to establish a life-long latency after a primary acute infection. In the case of HCMV, reactivation from latency can cause severe clinical effects in transplant and AIDS patients. Thus, it is critical to understand the mechanisms that regulate latency and reactivation as well as the cell types where it occurs (Reeves & Sinclair, 2008). Results from several studies suggest that myeloid progenitors (CD34+) are sites of latency for HCMV in vivo. The viral genome is maintained, as an episomal molecule, as these cells differentiate to monocytes. It is still unclear, however, why other lineages such as T cells or B cells which also arise from CD34+ progenitors do not carry HCMV genomes (Sinclair, 2008). Latency establishment and reactivation is closely related to the regulation of IE gene expression by repression or activation of the major immediate-early promoter region (MIEP) but it is not clear if expression of other genes may also be involved. An experimental latent model system

8

Chapter 1 based on infection of granulocyte-monocyte precursor cells (GM- Ps) permitted the identification of cytomegalovirus latency- expressed transcripts (CLTs) consisting of spliced and un-spliced RNAs mapped to both strands of the MIE region of the HCMV genome (Kondo et al. , 1996) and the UL111.5A transcript which encodes a viral homologue of interleukin-10 (vIL-10) (Jenkins et al. , 2004). Other transcripts shown to be expressed during latency in CD34+ progenitor cells were encoded by UL138 (Goodrum et al. , 2007) and LUNA (Bego et al. , 2005). Virus reactivation is associated with myeloid differentiation as viral lytic gene expression only occurs when these cells differentiate into macrophage or dendritic cell phenotypes (Reeves & Sinclair, 2008).

Experimental work on the different aspects of HCMV biology, such as latency and reactivation, has relied on human cell culture models due to the restricted species-specific host range of HCMV infection. There are, however, animal models such as murine CMV (MCMV), rat CMV, guinea pig CMV, and rhesus macaque CMV which are related to HCMV and naturally infect a laboratory animal host. The MCMV has been one of the most studied animal models both because of the close biological relation with HCMV and because of the selection of mutant strains of mice that facilitate studies in vivo involving cellular and immune pathways common to rodents and humans (Mocarski et al. , 2007).

9

Chapter 1

1.1.1.4. The UL24 gene family

The UL24 gene is located in the unique long segment of HSV-1 genome, overlapping the thymidine kinase gene (McGeoch et al. , 1988). It is conserved not only in all three subfamilies of human herpesviruses, but also in other mammalian, avian and reptilian herpesviruses, with exception of amphibians or fish (Table 2).

Of the core herpesviruses genes, UL24 is the only one that remains unassigned to any functional category (Davison et al. , 2002). Its universal presence in herpesviruses and lack of homology with cellular genes suggests that UL24 gene family has a relevant role in the viral life cycle and/or host evasion mechanisms.

The UL24 homologues are generally expressed with late kinetics, although HSV-1 UL24 has a complex transcription pattern with leaky-late kinetics since its expression is not completely dependent on viral replication (Pearson & Coen, 2002). The HSV-2 UL24 and HCMV UL76 homologues have unambiguously been identified as virion-associated proteins, and so they are found within the cell from the moment of infection (Hong-Yan et al. , 2001; Wang et al. , 2000). The alignment of the predicted amino acid sequences of UL24 homologues reveals five regions of strong sequence similarity (Jacobson et al. , 1989). Interestingly, a comparison of sequence profiles enriched by predicted secondary structure, has identified UL24 as a novel PD-(D/E)XK endonuclease belonging to a large superfamily of restriction endonuclease-like fold proteins (Knizewski et al. , 2006). To date, however, no nuclease activity has been reported for UL24 or any of its homologues.

10

Chapter 1

Subfamily Virus Gene

Human Herpesvirus 1 (HSV-1) UL24

alpha Human Herpesvirus 2 (HSV-2) UL24

Human Herpesvirus 3 (VZV) ORF35

Human Herpesvirus 5 (HCMV) UL76

beta Human Herpesvirus 6 U49

Human Herpesvirus 7 U49

Human Herpesvirus 4 (EBV) BXRF1 gamma Human Herpesvirus 8 (KHSV) ORF20

Table 2 . The human herpesvirus UL24 gene family

Deletion of the UL24 gene in HSV-1 resulted in a virus with significantly reduced plaque size and an associated decreased viral yield, suggesting that the UL24 function, although not essential, is important for virus growth, at least in cell culture (Jacobson et al. , 1998; Jacobson et al. , 1989). Similar results were obtained for HSV-2 UL24 (Blakeney et al. , 2005), VZV ORF35 (Ito et al. , 2005) and HCMV UL76 from AD169 strain (Yu et al. , 2003). Global functional analysis conducted using HCMV Towne complete genome indicated that UL76 is an essential gene for viral replication of this viral strain (Dunn et al. , 2003). This result, however, may be due to the impact of UL76 mutation on the overlapping UL77 gene as a recent study demonstrated that UL76 from Towne strain is not essential for viral growth, although it showed a slower replication cycle at low multiplicities of infection. Interestingly, the same study identified UL76 as a gene regulator of UL77 expression, possibly to control appropriate time and

11

Chapter 1 concentration of this viral gene for efficient viral replication (Isomura et al. , 2010).

Further deletion studies revealed that the absence of UL24 from HSV-1, HSV-2 and VZV leads to a syncytial plaque phenotype, similar to that observed for other HSV-1 viral proteins, suggesting that UL24, like UL20 and gK, may have a role in viral egress (Blakeney et al. , 2005; Ito et al. , 2005; Pearson & Coen, 2002), although the possible involvement of UL24 in assembly and egress of virus particles remains to be explored.

During viral infection, UL24 homologues are detected predominantly in the nucleus, and transiently localize in the nucleoli (Hong-Yan et al. , 2001; Nascimento & Parkhouse, 2007; Pearson & Coen, 2002; Wang et al. , 2000). Several studies demonstrated that UL24 is responsible for nucleolin and B23 redistribution in the nucleus during HSV-1 infection (Lymberopoulos & Pearson, 2007). Thus, it is possible that UL24 plays a role in nuclear egress through its impact on nucleoli, although how nucleolin affects HSV-1 nuclear egress is still unclear. The fact that deletion of conserved homology domains of UL24, including the putative endonuclease motifs, resulted in loss of nucleolin and B23 dispersal activity, suggests that this function may be shared among all herpesviruses and must be relevant for the viral life cycle (Bertrand et al. , 2010; Bertrand & Pearson, 2008; Lymberopoulos et al. , 2011).

In addition to dispersion of nucleolar proteins, the UL24 gene family modulates the cell cycle. The first results that demonstrated UL24- mediated cell cycle manipulation were obtained for the UL24 homologue from MHV-68, the ORF20 protein (Nascimento & Parkhouse, 2007). This virus is a particularly useful model for the

12

Chapter 1 study of herpesviruses biology in vivo . Similar to the human homologues, ORF20 is located in the nucleus and, when transiently expressed in human and murine cell lines, it induces cell cycle arrest at the G2/M phase, followed by apoptosis at later time points. During the G2 phase, the cyclin B/Cdc2 complex is inactive as the Cdc2 protein is in the inhibitory phosphorylated form. Consistent with the observed G2 arrest, cells expressing MHV-68 ORF20 showed an increased phosphoryation of Cdc2 at the inhibitory Tyr15 site and a consequent inactivation of Cdc2-cyclin B complex was demonstrated (Nascimento & Parkhouse, 2007). As observed for MHV-68 ORF20, the UL24 homologues from human herpesviruses representative of each subfamily (HSV-1 UL24, HCMV UL76 and KSHV ORF20) also induced cell cycle arrest followed by apoptosis through the same mechanism (Nascimento et al. , 2009). The precise mechanism of cell cycle arrest induced by UL24 homologues remain to be clarified. An interesting and possible explanation is the recent report that HCMV UL76 induces chromosomal aberrations and DNA damage (Siew et al. , 2009).

The role of UL24 homologues in viral pathogenesis in vivo, was explored using two independent ORF20 deletion mutant MHV-68 viruses. After intranasal inoculation of mice, MHV-68 replicates in lung epithelial cells (lytic phase) before establishing latency in the spleen. Mice infected with both ORF20 mutant viruses presented a delay in the clearance of the virus from the lung. In contrast to the extended acute phase of ORF20 mutant viruses, no major difference was found in the reactivation from latency of mutant virus compared to MHV-68 wild type infection (Nascimento et al. , 2011). The fundamental cause of the extended viraemia in mice infected with ORF20 mutant viruses remains to be elucidated. It

13

Chapter 1 would be important to analyze the ORF20 mutant MHV-68 pathogenesis in its natural host, the wood mice (members of the genus Apodemus ) (Ehlers et al. , 2007) as a recent comparative analysis revealed that MHV-68 infection of BALB/c (M. musculus ) and laboratory-bred wood mice are markedly different. The chemokine binding protein M3 of MHV-68 modulates the host response to infection in the natural host, although it has no effect during infection of Mus musculus -derived strains via the respiratory tract (Hughes et al. , 2010).

1.2. HCMV-host interaction

1.2.1. Virus modulation of cell cycle and DNA Damage response

The eukaryotic cell cycle is operationally divided into four phases: G1, the first “gap” during which cells organize themselves prior to DNA replication, S phase, where DNA synthesis occurs, G2, the second “gap” during which the cell checks the fidelity and organization of its replication prior to the process of mitosis (M) and the associated cell division. Quiescent cells, which are metabolically active but not dividing, are in the G0 phase, outside the cell cycle. The DNA damage-induced cell cycle checkpoints, G1/S, intra-S and G2/M, are critical points in the cell cycle that monitor the integrity of the genome, leading to repair or programmed cell death if damage is detected. If DNA damage is minor, DNA repair may occur without activating the checkpoint. On the other hand, if DNA damage is extensive, cells will undergo apoptosis and so the integrity of the genome is maintained (Abraham, 2001). DNA double strand breaks can be produced by endogenous sources such as reactive oxygen species generated

14

Chapter 1 during cellular metabolism and collapsed replication forks, or from exogenous sources including ionizing radiation or chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy (e.g. etoposide, camptothecin) (Shrivastav et al. , 2008). The DNA damage sensor molecules (e.g. RPA, MRN) that activate the checkpoints appear to be shared by all the three pathways, or at least, to play a primary sensor role in one pathway and a secondary role in the others. The same is observed for the signal transducers molecules, such as protein kinases (ATM and ATR) and phosphatases, which are shared by the different checkpoints to varying degrees. The specificity of the checkpoint is due to the effector proteins, which inhibit phase transition (Sancar et al. , 2004). Following cell cycle arrest, mammalian cells possess two main pathways for the repair of DNA double-strand breaks: homologous recombination (HR) and non-homologous end-joining (NHEJ). Double strand breaks, such as those produced by nucleases or ionizing radiation can be repaired by both pathways, while double strand breaks that result from stalled replication forks are repaired primarily by HR. During the cell cycle there is a shift in the balance between NHEJ and HR. In general, HR is considered to be a more accurate repair mechanism because broken ends use homologous sequences in the genome (sister chromatids or homologous chromosomes, for example) to prime repair synthesis. Since template accessibility influences HR efficiency, there is an up- regulation of HR during S and G2 phases of the cell cycle, when sister chromatids are available. During G1 and early S phases, NHEJ is the predominant repair pathway (Cann & Hicks, 2007). The regulatory factors that modulate this balance, in addition to the availability of homologous repair templates already mentioned,

15

Chapter 1 include expression and phosphorylation of repair proteins and chromatin modulation of repair factor accessibility (Shrivastav et al. , 2008).

Manipulation of the host cell cycle is a frequent virus strategy for host evasion, presumably in order to achieve a cellular environment favorable for their replication. For example, small DNA viruses capable of infecting non-dividing cells induce S phase in order to activate and utilize the host DNA replication machinery (Sato & Tsurumi, 2010). In contrast, herpesviruses encode their own DNA polymerase and accessory factors, and do not require the environment of an S phase for viral replication (Flemington, 2001; Lu & Shenk, 1999; Song et al. , 2000; Sullivan & Pipas, 2002; Sunil-Chandra et al. , 1992). An important aspect of the effects of viruses on cell cycle dynamics are the consequences for neoplastic transformation. This has been a major area of research, as it offers a rational approach to the control of virus associated cancers. Examples of cell cycle regulation by herpesviruses can be found in all three α-, β-, and γ-herpesvirinae subfamilies. Herpes simplex virus type 1 (HSV-1) infection disruption of cell cycle progression depends on the cell cycle phase (Ehmann et al. , 2000; Song et al. , 2000). Similar to HSV-1, several studies demonstrated that HCMV infection leads to drastic and temporally coordinated alterations in the expression of host cell regulatory proteins, such as cyclins, resulting in cell cycle arrest at more than one phase (Bresnahan et al. , 1996; Dittmer & Mocarski, 1997; Jault et al. , 1995; Lu & Shenk, 1996). The cell cycle arrest was independent of the cell cycle phase (G0, G1, and S) at the moment of infection with HCMV (Salvant et al. , 1998). The immediate-early proteins IE1/IE2 and UL69 have been described as being able to block cell cycle in G1

16

Chapter 1 suggesting that they may have a role in HCMV manipulation of cell cycle progression (Castillo et al. , 2005; Lu & Shenk, 1996; Wiebusch & Hagemeier, 1999). Recent studies with HHV-6A, one of the less studied β- herpesviruses, demonstrated that infection of T cells with HHV-6A results in cell cycle arrest at the G2/M phase. In this case, G2 arrest may serve to block the clonal expansion and proliferation of HHV-6A specific T cells to maintain immune suppression and evade the antiviral immune response (Li et al. , 2011). A common strategy of several gamma-herpesviruses is the expression of a viral cyclin (v-cyclin) with homology to the cellular D-type cyclin (Upton et al. , 2005; van Dyk et al. , 1999; Verschuren et al. , 2004). Reminiscent of cellular cyclins, v-cyclin interacts with, and thus activates, the Cdk4 and Cdk6 kinases. Although the v- cyclin is functionally similar to cellular cyclin, it is resistant to inhibition by p21 and p16 and it is not regulated by the cyclin- dependent kinases (Cdks) (Direkze & Laman, 2004).

The regulation of cell cycle by viruses is closely related to activation of the DNA Damage response, including double strand break repair pathways. Viral infection confronts the host cell with large amounts of exogenous genetic material that might be recognized as abnormal and damaged DNA and so precipitate the premature apoptosis of the virus infected cells (Weitzman et al. , 2004). For example, during replication, herpesvirus DNA is synthesized in a rolling-circle manner to produce head-to-tail concatemers that are subsequently cleaved into unit-length genomes. These too may be recognized as double strand breaks and trigger a DNA damage response (McVoy & Adler, 1994). Thus, in order to establish a productive infection, it is essential that

17

Chapter 1 viruses defend themselves from the host cell DNA damage response machinery. Paradoxically, recent reports indicate that such cellular responses may have a beneficial role in viral replication (Luo et al. , 2011). Activation of the DNA damage response during HCMV replication is indicated by the phosphorylation of ATM and H2A.X and the downstream proteins Chk2 and p53. However, during HCMV infection the localization of various checkpoint proteins, normally organized near the site of damage, is altered, inhibiting their normal function. Thus, HCMV activates a DNA damage response, but escapes its consequences due to “mislocalization” of checkpoint proteins (Gaspar & Shenk, 2006). Although previously it was concluded that ATM is not relevant for HCMV replication (Luo et al. , 2007), recent results indicate that the DNA damage response mediated by E2F1 transcription factor contributes to replication of HCMV (E et al. , 2011). Furthermore, infection with HCMV stimulates the homology-directed repair pathway, which might indicate that HCMV, like HSV-1, exploits the cellular proteins involved in cellular DNA repair to enhance virus genome replication and fidelity (Kulkarni & Fortunato, 2011). It is still not clear if virus- induced DNA damage involves the recognition of existing double strand breaks. In the case of HCMV infection, although the mechanism of E2F1-induced DNA damage response is still unknown, the inactivation of Rb and subsequent deregulation of E2F1 results in double strand breaks in human fibroblasts (Pickering & Kowalik, 2006). It is also known that infection of HCMV results in double strand breaks on chromosome 1 (Fortunato et al. , 2000), but it is not clear if this is sufficient to activate the observed DNA damage response. Interestingly, prolonged binding of DNA repair factors to chromatin can elicit

18

Chapter 1

DNA damage response in an ATM- and DNA-PK-dependent manner in the absence of DNA lesions (Soutoglou & Misteli, 2008). Thus, it is possible that the trigger of herpesvirus-induced DNA damage response is not the recognition of viral DNA as double strand breaks or actual damage to DNA, but it is the recruitment of DNA damage repair factors observed during viral infection.

The beneficial role of activation of DNA damage in viral replication is not restricted to herpesviruses as it has also been observed for several other viruses. Simian virus 40 (SV40) replication is dependent of ATM-mediated phosphorylation of large tumour antigen (LTag), an essential viral protein involved in viral replication (Shi et al. , 2005). Human papillomavirus (HPV) infection also induces an ATM response in both undifferentiated and differentiated cells. Importantly, ATM kinase activity is required for viral genome amplification in differentiating cells, but not for episome maintenance in undifferentiated cells. This suggests that activation of the DNA damage signaling response by HPV is tailored to different requirements, depending on the differentiation stage of the host cell (Moody & Laimins, 2009). Adenovirus, however, has evolved mechanisms to inhibit DNA repair during infection, by degradation and mislocalization of the Mre11–Rad50– NBS1 complex, thus preventing activation of DNA damage checkpoints and viral DNA concatemerization. The model proposed is that the DNA damage response results in the masking of the origins of adenovirus DNA replication such that viral replication proteins are unable to gain access (Stracker et al. , 2002).

Understanding the interaction between the DNA damage response machinery and virus infections will not only provide insights into

19

Chapter 1 viral pathology and persistence, but also new ideas for the development of antiviral and anti-tumour drugs. Moreover, the study of virus host evasion mechanisms can provide new tools to study recognition and repair of damaged DNA by cellular machinery.

1.2.2. Host immune response to virus infection

Host cells have at least three layers of defense mechanisms which include autonomous innate mechanisms, such as RNA interference and cytidine deamination (these will not be discussed further), nonautonomous innate mechanisms induced by exposure to viruses and other stimuli such as interferon and proinflammatory cytokines and adaptive (or acquired) immunity resulting in an antigen-specific response via antibodies and lymphocytes. The aim of these systems is to clear the viruses and virus-infected cells, or at least to limit the pathogenicity and viral spread. To achieve this goal, there is a significant interplay between them, although they present significant differences related with their rapidity, specificity, persistence, evolutionary antiquity and consequences for the infected cell (Biron & Sen, 2007).

The non-autonomous innate immune system is mediated by cytokines such as interferon and chemokines, local sentinel cells such as dendritic cells and macrophages, the complement system, and natural killer (NK) cells. The primary burst of cytokines that are released from infected cells, amplifying the initial response to virus infection, involves dendritic cells, macrophages, neutrophils and other granulocytic white blood cells (Malmgaard, 2004).

20

Chapter 1

Innate immune recognition of viruses as foreign, and thus potentially dangerous, is performed by a limited number of germline-encoded pattern-recognition receptors (PRRs). The host cell PRRs immediately distinguish self molecules from viruses through recognition of microbial components, known as pathogen- associated molecular patterns (PAMPs), which are expressed constitutively by pathogens. Toll ‑like receptors (TLRs), membrane ‑bound receptors that are localized in the plasma membrane and endosomes, are the best characterized PRRs. Of particular interest for virus infection is the endossomal TLR3, which recognizes viral double stranded RNA, and TLR7, TLR8 and TLR9 which recognise viral DNA. Intracellular PRRs detect pathogen nucleic acids in the cytoplasm. RNA is recognized by the RIG ‑I‑like receptors (RLRs) retinoic acid ‑inducible gene I (RIG ‑I) and melanoma differentiation ‑associated gene 5 (MDA5). Intracellular DNA sensors include DNA-dependent activator of IFN ‑regulatory factors (DAI), absent in melanoma 2 (AIM2), RNA polymerase III, leucine ‑rich repeat flightless interacting protein 1 (LRRFIP1) and, most recently, IFN γ‑inducible protein 16 (IFI16). Activation of TLRs and intracellular nucleic acid sensors results in the induction of signalling pathways that lead to the expression of proteins with pro ‑inflammatory and microbicidal activities, including cytokines and type I IFNs (IFN α and IFN β) (Mogensen, 2009; Takeuchi & Akira, 2010).

Interferons are a group of cytokines with a variety of biological functions, including modulation of the immune system, regulation of apoptosis, inhibition of proliferation, induction of differentiation, and inhibition of angiogenesis (Uddin & Platanias, 2004). The

21

Chapter 1 importance of the interferon response against viral infections has been emphasized by the increased susceptibility to virus infection of mice deficient for different components of the IFN system (Arnheiter et al. , 1996; Hefti et al. , 1999).

Interferons are a large family of related cytokines which include type I IFNs, type II IFNs and type III IFN. The IFNs that are induced directly in response to virus infection are IFN-α and –β, which bind to a widely distributed receptor. Interferon-γ, a type II IFN and commonly called “immune IFN”, on the other hand, binds to a different cell-surface receptor and is only secreted by activated T cells and NK cells (Farrar & Schreiber, 1993). The type III IFNs are also secreted after viral infection and following stimulation of key molecules involved in type I IFN induction, such as IRF-3, RIG-I, and NF-кB (Onoguchi et al. , 2007). Once secreted, Type III IFNs bind to specific cellular receptors eliciting an antiviral response similar to IFN α/β, with the induction of transcription of hundreds of inferferon stimulated genes (ISGs) (Sommereyns et al. , 2008).

One of the major functions of interferon is the induction of an anti- viral state in cells infected by viruses, characterized by the expression of genes induced by interferon in order to limit virus replication and subsequent spread to neighbouring cells. The ISGs are crucial components of the interferon responses as they set up the antiviral, anti-proliferative and immunoregulatory state in the host cells. The best characterized IFN inducible components are the enzymes dsRNA-dependent protein kinase (PKR), 2’,5’- oligoadenylate synthetase (2’5’OAS), and Mx proteins (García et al. , 2006; Haller et al. , 2007; Silverman, 2007).

22

Chapter 1

The innate immunity mechanisms are clearly ineffective in the clearance of the herpesviruses and in preventing the consequent establishment of latency, although it controls the viral replication leading to a generally asymptomatic infection. In the case of HCMV, the innate immune response initiated after the binding and entry of the virus into the cell involves the activation of IRF-3 that will lead to the production of type I interferon via the DNA sensor DAI (DeFilippis et al. , 2010a) and pro-inflammatory cytokines by CD14 and TLR2 recognition (Compton et al. , 2003). The possible involvement of structural viral proteins in the induction of innate immune responses was confirmed by the identification of the HCMV envelope glycoproteins gB and gH as proteins essential for TLR2 activation (Boehme et al. , 2006). Recently, however, it has been demonstrated that glycoprotein B is not essential for the activation of IRF-3-dependent innate immune response. In fact, IRF3 signaling activated by HCMV infection is very similar to the pathway activated by cytoplasmic double-stranded DNA (DeFilippis et al. , 2010b). An important component of the innate immune response to viral infections is the natural killer (NK) cell population. The importance of this subset of cells in cytomegalovirus immunity is evident in the MCMV infection of mice but there is limited evidence in the case of HCMV infection. The fact that HCMV encodes several proteins that prevent NK cell activation by different mechanisms underlines the conclusion that NK cells have an important role in the immune response to HCMV infection as well (Jackson et al. , 2011). In contrast to innate immunity, the adaptive immune response is characterized by the specificity of adaptive immune cells for a particular foreign viral antigen, and the capacity of this system to recognize and respond to a great diversity of viruses. The cells of

23

Chapter 1 the adaptive immune system, B lymphocytes and T lymphocytes, are activated by the recognition of viral constituents, through the binding to highly specific cell surface recognition receptors. These receptors are specific to the constituents, designated by antigens, of that particular virus. Importantly, the adaptive immune system adapts to repeated exposure to a particular virus or viral protein, resulting in a more rapid response of higher magnitude by the specific adaptive immune cells during a secondary infection with that virus. This phenomenon is called immunological memory and it is the basis for vaccination (Braciale et al. , 2007).

The B and T lymphocytes differ in their recognition and effector mechanisms. The B lymphocyte receptor is a clonally expressed immunoglobulin (Ig) molecule capable of binding free (extracellular) virions in a highly specific matter leading to virus neutralization and elimination (humoral response). Upon interaction with the corresponding antigen, in this case, the virus, the cell is activated to divide and differentiate into a plasma cell secreting antibodies with the identical specificity of the receptor Ig. Antibodies that interfere with virus entry into cells are called neutralizing antibodies. Until recently, it was thought that this extracellular activity of antibodies was the only relevant contribution to serological antibody-mediated immunity. Recently, however, in a very interesting paper, it was demonstrated that non-enveloped virus-antibody complexes can be internalized and signal proteosome mediated degradation of the virus particle, and thus potentially contributing to anti-virus immunity (Mallery et al. , 2010). Antiviral B-lymphocyte responses have a critical role not only in clearing the virus during infection, but also in preventing or limiting re-infection and in vaccination against a specific virus. The humoral

24

Chapter 1 response to primary HCMV infection includes the production of specific antibodies for several structural and non-structural proteins, as well as for envelope glycoproteins (Landini & Michelson, 1988) and these play an important role in preventing transmission to the fetus during pregnancy (Fowler et al. , 1992). Similarly, T lymphocytes maintain their antigen-specific receptors on the cell surface. Upon activation T cells are important in the recognition and elimination of virus-infected cells. The T lymphocytes antigen receptors recognize processed peptide fragments of intracellular viral proteins presented on the infected cell surface by the major histocompatibility complex (MHC) class I. Alternatively, antigens may be internalized by antigen presenting cells, such as dendritic cells and macrophages. After intracellular degradation, the resulting peptides are incorporated into the MHC class II molecules which are transported to the surface of the antigen presenting cell and are recognized by CD4+ T cells, thus leading to their activation and participation in both serological and CD8+ cytotoxic immune responses.

Activated CD8+ T lymphocytes limit the spread of infection by killing virus-infected cells through direct cell-to-cell contact and by the release of soluble mediators which include cytokines such as interferon gamma and tumor necrosis factor alpha (TNF-α). The activated T lymphocytes, both CD4+ and CD8+, through these and other soluble mediators, recruit and coordinate the response of the innate immune cells which, in turn, act to clear infection. The cellular immune response to cytomegaloviruses includes CD8+, CD4+ and gamma delta T lymphocytes. Studies based on the murine MCMV model and transplantation patients revealed that HCMV specific CD8+ T cells are crucial in the immune response to

25

Chapter 1

HCMV. Similar studies are limited during primary infection, however, partially because it is frequently asymptomatic (Crough & Khanna, 2009). Decades of research identified several HCMV proteins that are targets of the CD8+ T cell response to HCMV. However, the majority of the studies on the immunobiology of the CD8+ T cell response in primary infection and the generation of long term memory have been using the first identified viral targets, namely pp65 and IE proteins (Borysiewicz et al. , 1988; McLaughlin- Taylor et al. , 1994). Early studies with MCMV infected mice showed that CD4+ T cell are also an important component of the cellular immunity response to cytomegaloviruses as long term depletion of the whole CD4+ T cell compartment resulted in persistent virus replication at specific sites (Jonji ć et al. , 1989). Furthermore, the maintenance of HCMV specific CD8+ T cell infusions in bone marrow transplant patients was dependent on the presence of HCMV-specific CD4+ cells indicating a critical role of CD4+ helper T cells in the effective response of CD8+ T cells (Einsele et al. , 2002; Walter et al. , 1995). Finally, there is evidence that a subset of gamma delta ( γδ ) T cells (the minor V δ2 negative subpopulation) is expanded following HCMV reactivation in transplant patients and is able to mediate cytotoxicity of HCMV infected target cells (Knight et al. , 2010).

1.2.3. Virus modulation of immune system

Under the pressure of host immune defenses, viruses have evolved a large number of genes that act at several levels to counteract host cell biology and immune responses and so ensure their survival and replication culminating, in the case of

26

Chapter 1 herpesviruses, in the establishment of persistence during the host’s life time.

The interferon system is a powerful and first line of defence against virus infections, and so it is not surprising that viruses have evolved multiple means of down regulating the induction and impact of IFN responses. These include inhibiting IFN production, inhibiting the IFN-mediated signalling pathways, and blocking the action of IFN- induced enzymes with antiviral activity. The exact strategy exploited by a virus will presumably depend on the biology of the host-virus interaction and will be a major factor that will influence the pathogenesis of that virus infection (Randall & Goodbourn, 2008). Herpesviruses induce expression of type I IFN during the primary infection (Ankel et al. , 1998; Boehme et al. , 2004). Thus, it is not surprising that an effective evasion of these initial type I IFN responses is essential for virus replication and establishment of latency.

In the case of HCMV infection, only a small number of genes have been identified as modulators of the interferon response. The immediate-early proteins of HCMV are the obvious candidates, as they are the first genes being expressed, and indeed, the IE72 protein has been shown to play a role in inhibiting the antiviral state by binding to promyelocytic leukemia (PML) protein and disrupting PML-associated nuclear bodies (NBs) leading to the displacement of PML-NB associated proteins such as PML, Sp100 and Daxx (Ahn & Hayward, 1997; Korioth et al. , 1996). In addition, IE72 also binds to STAT2 and, to a lesser extent, to STAT1, thereby inhibiting the IFN signalling pathway (Huh et al. , 2008; Paulus et al. , 2006). The IE86 protein has been described as an inhibitor of IFN-β production by blocking NF κB (Taylor & Bresnahan, 2005).

27

Chapter 1

On the other hand, UL83 has been also shown to inhibit IFN-β production by inhibiting IRF-3 phosphorylation and translocation into the nucleus (Abate et al. , 2004). HCMV also encode two PKR antagonists, proteins IRS1 and TRS1 (Cassady, 2005).

Natural killer (NK) cells are crucial in controlling cytomegalovirus infections in both the human and the murine hosts. NK cells are inhibited by signals delivered via inhibitory receptors interacting with class I MHC molecules on the surface of target cells (Jackson et al. , 2011). Interestingly, cells infected with HCMV are remarkable resistant to NK cell-mediated cytolysis in vitro . Not surprisingly, several genes encoding proteins (UL16, UL18, UL40, UL83, UL141 and UL142) and one encoding a microRNA (miR-UL112) have been identified as capable of suppressing NK cell recognition through presentation of inhibitory signals or suppression of activating signals (Wilkinson et al. , 2008). One of the mechanisms of HCMV-mediated inhibitory receptor signalling is the expression of UL18 gene, a MHC class I homologue present at the cell surface where it binds the inhibitory NK cell receptor LILRB1 (LIR-1) inhibiting activation of NK cells (Prod'homme et al. , 2007). On the other hand, HCMV infection efficiently activates expression of ligands for the NK cell activating receptor NKG2D. However, at least two proteins, UL16 and UL142, and the miR-UL112 collaborate to suppress presentation of these ligands on the cell surface inhibiting NK cells activation (Wilkinson et al. , 2008).

In addition to mechanisms that interfere with MHC class I expression on the cell surface, such as removal of peptides or intracellular retention of the MHC complexes, HCMV also induces the degradation of MHC class I molecules by expressing US2 and US11 genes (Wiertz et al. , 1996a; Wiertz et al. , 1996b). In addition,

28

Chapter 1

HCMV and MCMV interfere with CD4+ T cell recognition of infected cells by inhibition of MHC class II expression (Heise et al. , 1998; Scholz et al. , 1992; Sedmak et al. , 1995). Moreover, it has been demonstrated that HCMV evolved different mechanisms to downregulate MHC class II molecules at early and late times after infection. The early inhibition is probably due to a structural component while the late effect was dependent on virus replication and expression of US1-US9 or US11 (Odeberg & Söderberg- Nauclér, 2001).

1.2.4. Virus modulation of chemokines

Chemokines are structurally related molecules that regulate the trafficking and activation of various types of leukocytes through the interaction with a subset of seven-transmembrane G protein- coupled receptor (GPCR) and thus may be important mediators of host defense mechanisms. Chemokines can be divided in three subfamilies: α-chemokines (C-X-C), which act primarily on neutrophils (chemotaxis), β-chemokines (C-C), which act on monocytes, lymphocytes, basophils and eosinophils (chemoattraction and degranulation), and γ-chemokines, which act upon lymphocytes (Zlotnik & Yoshie, 2000).

During the long parallel evolution with their hosts, viruses developed diverse mechanisms to modulate chemokines and their receptors in order to evade the control of the immune system. Large DNA viruses such as herpesviruses encode chemokine homologues which recruit leukocytes to facilitate its dissemination and growth, thus subverting the natural function of these molecules as modulators of immune response to viral infections; for example, the vCXC-1, encoded by UL146 gene from HCMV (Penfold et al. ,

29

Chapter 1

1999) and the vMIP-I (K6 gene) and vMIP-III (K4.1) from KSHV which show angiogenic activity (Boshoff et al. , 1997; Kledal et al. , 1997; Stine et al. , 2000). The chemokine homologues are not shared by closely related herpesviruses, suggesting that they may be related to specific aspects of pathogenesis. Other common evasion mechanism includes the inhibition of chemokine signaling by the secretion of soluble chemokine-binding proteins or by encoding GPCR homologues which mimic the functions of cellular GPCR (Alcami, 2003). A well studied example is the US28 gene from HCMV encoding a vGPCR for β-chemokines (Gao & Murphy, 1994). Constitutively activated, US28 protein can regulate the chemokine environment by sequestering CC chemokines and thus, depleting them from the medium (Bodaghi et al. , 1998). In addition, viruses hijack chemokine receptors as co-receptors for viral entry and intracellular signaling. For example, chemokine receptors CCR5 and CXCR4 are major HIV-1 co-receptors which mediate viral entry into susceptible cells (Deng et al. , 1996; Feng et al. , 1996). In addition to encoding viral homologues of cellular proteins involved in the signaling of chemokines, generally viruses are able to induce or inhibit expression of several chemokines. Interleukin-8 is an important example as it is induced by several viruses and it will be described in more detail.

1.2.4.1. IL-8

Interleukin-8 (IL-8) is a pro-inflammatory chemokine that attracts neutrophils, monocytes and cytotoxic T cells (Taub et al. , 1996) by interacting with the CXC chemokine receptors CXCR1 and CXCR2. Besides its relevant role in inflammation, IL-8 is also involved in

30

Chapter 1 angiogenesis (Koch et al. , 1992) and interferon-α inhibition (Khabar et al. , 1997). IL-8 expression is low or absent under normal conditions but highly inducible in vivo, as well as in a wide variety of cells in vitro, by a wide range of extracellular stimuli in addition to viruses, such as the proinflammatory cytokines IL-1 or TNF-α (Kasahara et al. , 1991) or by bacteria (Aihara et al. , 1997) and cell- stressing agents.

1.2.4.1.1. Gene regulation

Interleukin-8 plays a significant role in recruiting leukocytes at sites of acute inflammation; however, excessive amounts of locally produced IL-8 can have deleterious effects. Therefore, IL-8 gene expression is tightly controlled at several levels. It was previously described that the sequence spanning nucleotides 21 to 2133 within the 5’ flanking region of the IL-8 gene is essential and sufficient for transcriptional regulation of the gene. This promoter region contains an NF-kB element that is required for activation in the majority of cell types studied, as well as AP-1 and NF-IL-6 binding sites. The latter two sites may be dispensable for transcriptional activation in some cells but contribute to optimal IL-8 activation. This cooperation between different pathways and the close proximity of NF-kB, AP-1 and NF-IL-6 in IL-8 promoter suggests the formation of nucleoprotein complexes, named transcriptional enhanceosome, as in the case of interferon induction. The low levels of IL-8 expression in unstimulated cells are partly a result of transcriptional repression of the IL-8 promoter. Collectively, the results of several studies suggest a model, whereby IL-8 transcription is effectively repressed in unstimulated cells by a combination of three mechanisms involving deacetylation of histones, OCT-1 binding and active repression by NF-kB

31

Chapter 1 repression factor (NRF). The low amount of IL-8 found in unstimulated cells results not only from a repressed transcription but also due to mRNA instability. Expression of IL-8 is regulated post-transcriptionally by the p38 MAPK pathway that contributes to cytokines/stress-induced IL-8 by stabilizing the IL-8 mRNA (Hoffmann et al. , 2002). In conclusion, IL-8 production is actively repressed in the absence of external stimuli. During stimulation, maximal IL-8 amounts can be produced as a result of promoter derepression, activation of NF- kB and JNK pathways to induce transcription and the rapidly stabilization of the resulting mRNA by the p38 MAPK pathway. In this way, cells are able to rapidly increase and at the same time, regulate the amount of IL-8 secreted and thereby control the extent of leukocytes attracted to sites of tissue injury.

1.2.4.1.1.1. NF-KB

Nuclear factor-kB (NF-kB) consists of a family of transcription factors which in mammals include p65 (RelA), RelB, c-Rel, p105/p50 (NF-kB1), and p100/52 (NF-kB2). Each subunit can associate with each other to form distinct transcriptionally active homo- and hetero-dimeric complexes, with p50/p65 heterodimer being the most abundant and present in almost all cell types (Oeckinghaus & Ghosh, 2009). In unstimulated cells, NF-kB dimers are maintained in an inactive form in the cytoplasm through their interaction with IkB proteins. In certain cell types such as mature B cells, macrophages, neurons, vascular smooth muscle cells and a large number of tumor cells, however, NF-kB is constitutively active.

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Chapter 1

The NF-kB signaling pathway plays critical roles in inflammation, immunity, cell proliferation, differentiation, and survival as specific NF-kB binding sites are present in the promoters/enhancers of a large number of genes. Thus, not surprisingly, deregulation of NF- kB is associated with a considerable number of diseases (Perkins, 2007). The complexity of the NF-KB signaling is demonstrated by the diverse mechanisms of activation of NF-kB pathway. A simplified view of the two main pathways (canonical and noncanonical) is shown in Figure 1.1 and they will be briefly described.

Figure 1.1. Canonical and non-canonical signaling to NF-κB (Oeckinghaus & Ghosh, 2009).

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Chapter 1

1.2.4.1.1.2. NF-kB Canonical pathway Diverse inflammatory stimuli such as the cytokines TNF-α and IL– 1β activate the ubiquitous p65/p50 dimer through the ‘canonical’ or ‘classical’ NF-κB pathway (Fig. 1.1). Stimulation through TNF receptor (TNFR), IL–1β receptor (IL–1βR), or TLRs leads to the phosphorylation of I κB kinase (IKK) complex, composed of the two catalytic subunits, IKK α and IKK β, and the regulatory protein, IKK γ/NF-κB essential modulator (NEMO). The activated IKK complex phosphorylates IkB (inhibitor of NF-kB) proteins leading to their ubiquitination and degradation, thus releasing the NF-kB subunits to translocate to the nucleus and induce transcription of target genes. In most cell types, activation of the canonical pathway results in the nuclear localization of NF-kB dimers within minutes. Regulation of NF-kB signaling involves a series of post- translational modifications of the NF-kB subunits in addition to other proteins of the pathway (Oeckinghaus & Ghosh, 2009; Perkins & Gilmore, 2006).

1.2.4.1.1.3. NF-kB Non-Canonical pathway

Stimulation of the CD40 and lymphotoxin-β receptors on the other hand activates the ‘non-canonical’ (or alternative) NF-kB pathway (Fig 1.1). In contrast to the canonical pathway, there is activation of NF-kB-inducing kinase (NIK), which is followed by IKK α dimer activation. The p100 (NF-kB2) NF-kB subunit is then phosphorylated, inducing its proteolytic processing to p52. The p100 subunit has the inhibitory function of a cytoplasmic localized IkB protein since it contains the ankyrin repeats found in IkB proteins. Processing of p100 to p52 results in activation of NF-kB complexes containing this subunit, which generally consist of

34

Chapter 1 p52/RelB heterodimers. As different NF-kB dimers combinations have overlapping but distinct DNA-binding specificities, stimulation of both the canonical and noncanonical pathways lead to activation of a different spectrum of genes promoters and enhancers (Perkins & Gilmore, 2006).

1.2.4.1.1.4. NF-kB activation by genotoxic stress

In the canonical and non-canonical pathways, activation results from membrane-associated receptors stimulation. In recent years however, a different pathway activated by genotoxic stimuli, such as ionizing radiation or some chemotherapeutic drugs as etoposide, has been characterized. In this case, the activation signal comes from the nucleus although it converges in IKK complex activation in the cytoplasm as the canonical pathway (Fig. 1.2). Genotoxic stress induces, in parallel, activation of ATM and translocation of NEMO to the nucleus, where it is sumoylated by a mechanism dependent on PARP1, PIASy and Ubc9. NEMO sumoylation is followed by phosphorylation and mono-ubiquitylation in an ATM-dependent manner, leading to the nuclear export of NEMO as a complex with ATM. The activation of the IKK complex in the cytoplasm by the NEMO–ATM complex requires the associated protein ELK (a protein that is rich in glutamate (E), leucine (L), lysine (K) and serine (S)) (Janssens & Tschopp, 2006; Miyamoto, 2011). The mechanism underlying activation by genotoxic stress is still not completely understood but it is a clear example of how induction of NF-kB pathway can be integrated with parallel signaling pathways, in this case, DNA Damage response (Perkins, 2007).

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Figure 1.2. Activation of NF-κB pathway by genotoxic stress. Polyubiquitin is represented in repeated yellow units, phosphate is shown in orange oval with “P” and SUMOylation is shown in purple circle with “S” (adapted from Miyamoto, 2011).

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1.2.4.1.2. IL-8 and HCMV

It has been previously described that IL-8 enhances HCMV replication and virus production at 5 and 7 days after the infection, without initiating virus production earlier. An increase in IL-8 receptor was also observed in HCMV-infected cells, suggesting that HCMV infection may alter the responsiveness of cells to IL-8 by induction of IL-8 receptor expression, and in this way, modulate the intracellular environment to be more favorable for viral replication (Murayama et al. , 1994). In addition, HCMV infection increased the levels of IL-8 protein in human lung fibroblasts, astrocytoma cells, endothelial cells, peripheral blood mononuclear cells and a human monocytic cell line (THP-1) (Craigen et al. , 1997; Grundy et al. , 1998; Murayama et al. , 1997). Infection with high passage laboratory HCMV strains (AD169, Towne, Davis) or low passage clinical strains (Toledo and ClF isolates) showed that all virus strains significantly increase IL-8 production compared with uninfected cells (Craigen et al. , 1997).

Deletion mutant analysis of IL-8 promoter sequence demonstrated that NF-kB and AP-1 binding sites are required for IL-8 expression induced by HCMV, suggesting a critical role for both transcription factors (Murayama et al. , 1997). The mechanism of HCMV-induced IL-8, however, is still not clear. The only HCMV gene that has been described as IL-8 activator is the Immediate Early 1 (IE1) gene in which induction of IL-8 results from activation of both AP-1 and NF- kB pathways, consistent with previous studies with HCMV infection. Importantly, studies using ISIS 2922, an antisense oligonucleotide which blocks the production of IE proteins, only partly inhibited HCMV-induced IL-8 expression at the transcription and protein

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Chapter 1 levels, suggesting the involvement of one or more viral proteins (Cinatl et al. , 2000; Cinatl et al. , 1999). The IL-8 induced by HCMV infection is functional since IL-8 in supernatants from HCMV-infected cells significantly enhanced neutrophil transendothelial migration compared to those from uninfected cells. Moreover, infected endothelial cells were able to transmit the virus to neutrophils by both cell co-culture and neutrophil transmigration assays. Thus, a possible model is that HCMV-infected endothelial cells, in addition to other cells in the tissues, such as fibroblasts, recruit neutrophils to sites of infection by the secretion of IL-8. The infected endothelial cells then transmit the virus to the neutrophils, which disseminate the virus throughout the body via the bloodstream (Craigen et al. , 1997; Grundy et al. , 1998). Moreover, induction of IL-8 can also be considered to be a strategy of immune response evasion through disruption of normal cell migration patterns or the preferential attraction of leukocytes that cannot efficiently clear the virus (Penfold et al. , 1999). Interestingly, HCMV encodes two genes, UL146 and UL147 which are homologues to CXC chemokines. The UL146 gene encodes the protein vCXCL1 which functions as a selective agonist for CXCR2 and, although with lower affinity and potency, for CXCR1 (Lüttichau, 2010; Penfold et al. , 1999). The gene product of UL147 has not yet been characterized. The presence of a viral gene that mimics the effect of IL-8 in neutrophil chemotaxis reinforces the importance of neutrophils in the pathogenesis of HCMV. Thus, the absence of UL146 in HCMV AD169 strain or sequence divergence observed in several clinical strains may contribute to the observed attenuation of their associated pathogenesis. Surprisingly, recent studies have identified several HCMV proteins that down-regulate the production of proinflammatory cytokines

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Chapter 1 such as IL-8. A recent example is HCMV UL7, a homologue of the SLAM-family receptor CD229, which impairs the production of IL-8, among other proinflammatory cytokines, in different cell types (Engel et al. , 2011). Thus, HCMV may have evolved different mechanisms to induce IL-8 for its benefit, on the other hand, whilst also evolving genes that limit its upregulation, thus preventing excessive inflammation and consequent deleterious effects. Another interesting effect of IL-8 related to viral infections is the reduction of antiviral activity of IFN α observed in vitro (Khabar et al. , 1997). In summary, IL-8 induced by HCMV may aggravate HCMV infection by enhancing virus replication, virion production and viral dissemination due to neutrophil chemotaxis, in addition to inhibiting the antiviral activities of IFN. Thus, IL-8 might be a novel target for HCMV infection and associated diseases therapy.

1.3. Aim of the project

The importance of herpesviruses is evident by their prevalence in the human population and the diverse range of diseases that they provoke. They are among the most persistent of all pathogens, providing a primary example of how viruses successfully evade the immune system and manipulate cellular biology. One promising approach for the development of novel anti-viral strategies is to study viral proteins involved in host evasion. A large number of these proteins have already been identified and characterized through their homology with cellular proteins. There are, however, evasion proteins encoded by genes without sequence homology to cellular genes. In these cases, the viral protein function can only be accessed by functional assays or sophisticated structural assays,

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Chapter 1 such as x-ray crystallography. The UL24 gene family is one of the approximately 40 core genes that are conserved in all three herpesviruses subfamilies and the only one which still has no assigned function. Previously, functional assays demonstrated that all homologues of UL24 gene family induce cell cycle arrest (Nascimento et al. , 2009; Nascimento & Parkhouse, 2007), confirming that genes conserved in all herpesviruses are likely to manipulate conserved cellular pathways in a similar manner in all herpesviruses. Other strategies, on the other hand, may be restricted to one subfamily or species, with a function related to a more restricted and specific aspect of the virus life cycle. In this work, microarray analysis of cells expressing the HCMV UL24 homologue (UL76) gave new insight into herpesviruses host evasion strategies. Specifically, the aim of this project is to identify and characterize a new function for UL76, the UL24 homologue from HCMV, consisting in the induction of IL-8 and its impact during HCMV infection.

1.4. References

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CHAPTER 2

HUMAN CYTOMEGALOVIRUS UL76 PROTEIN

INDUCES IL-8 EXPRESSION

Chapter 2

2.1. Summary

Microarray analysis was performed to identify intracellular signaling pathways that are modulated by members of conserved, non- assigned UL24 gene family, specifically, ORF20 from MHV-68 and UL76 from HCMV. This work is focused on expression of IL-8, which was up-regulated in cells expressing UL76, but not ORF20. The microarray results were confirmed by reverse transcriptase PCR. Induction of IL-8 by the UL76 gene was demonstrated at the transcriptional level by luciferase assays using a reporter construct containing the IL-8 promoter, and at the protein level by the demonstration of increased levels of secreted IL-8 protein by ELISA. In summary, a new function of UL76, the stimulation of IL-8 expression, has been described.

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2.2. Introduction

Herpesviruses are a large group of double stranded DNA viruses, widely distributed and of serious medical and veterinary importance. Their life cycle is characterized by an acute phase of infection in epithelial cells followed by the establishment of persistence in a variety of cell types, depending on the virus subfamily (Pellett & Roizman, 2007). Thus, not surprisingly, herpesviruses have evolved a wide repertoire of host evasion genes that impact on both innate and adaptive immune response and so modulate the cellular environment to optimize viral replication, persistence and propagation.

The typical herpesvirus life cycle, with the two very different and sequential phases of acute and persistent lifestyles, is a challenge for the development of a global antiviral therapy or protective vaccines. One promising approach is to explore new viral targets conserved in all herpesviruses. The majority of conserved herpesvirus protein families with cellular homology have been already identified and characterized. There are, however, potential host evasion proteins encoded by genes without sequence homology to cellular genes (Holzerlandt et al. , 2002). In these cases, the viral protein function can only be determined by functional assays or structural assays, such as x-ray crystallography (Cooray et al. , 2007).

The UL24 gene family is conserved not only in all three subfamilies of human herpesviruses, but also in other mammalian, avian and reptilian herpesviruses. Interestingly, of the core herpesviruses genes, UL24 is the only one that remains unassigned to any functional category (Davison et al. , 2002). Its universal presence in

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Chapter 2 herpesviruses and lack of homology with cellular genes, however, suggests that UL24 gene family may have a relevant role in the viral life cycle and/or host evasion mechanisms. Previously, our group demonstrated that the UL24 human and murine homologues induce cell cycle arrest followed by apoptosis, through the inhibition of the mitotic Cdc2-cyclin B complex (Nascimento et al. , 2009; Nascimento & Parkhouse, 2007). Although a recent report showed that the UL24 homologue from human cytomegalovirus (UL76) induces chromosomal aberrations and DNA breaks (Siew et al. , 2009), suggesting the involvement of activation of DNA damage checkpoint, the precise mechanism of cell cycle arrest induced by the UL24 homologues remains to be clarified.

In this chapter, a preliminary exploration with DNA microarrays was done in order to define the impact of ORF20 and UL76, the UL24 homologues from murine herpesvirus 68 (MHV-68) and human cytomegalovirus (HCMV), respectively, on the cellular transcription profile. The development of DNA microarrays permits a global analysis of different intracellular signaling pathways required for the study of complex functional mechanisms. Microarray technology has several applications that are already routine, such as transcript profiling and genotyping, whereas others, such as genome-wide epigenetic analysis and ChIP-on-chip synthesis, are relatively new (Hoheisel, 2006). This work focused on the observation that the UL76 gene of HCMV promoted a very significant upregulation of interleukin-8 (IL-8).

The pro-inflammatory chemokine IL-8 attracts primarily neutrophils, but also monocytes and cytotoxic T cells, by interacting with the CXC chemokine receptors CXCR1 and CXCR2 (Baggiolini & Clark- Lewis, 1992). Expression of IL-8 is induced by a wide range of

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Chapter 2 extracellular stimuli such as the proinflammatory cytokine IL-1 or tumor necrosis factor (TNF α) (Kasahara et al. , 1991), cell-stressing agents (DeForge et al. , 1993), bacteria (Aihara et al. , 1997) and virus (Cota et al. , 2000; Medin & Rothman, 2006). Importantly, and of immediate relevance, it has been demonstrated that infection with HCMV induces the expression of IL-8 protein in different cell types (Craigen et al. , 1997; Murayama et al. , 1997), and this correlates with the observation that IL-8 enhances HCMV replication and virion production (Murayama et al. , 1994). Thus, the aim of this chapter is to unequivocally confirm that the HCMV gene, UL76, induces IL-8 expression at transcriptional and protein secretion level.

2.3. Materials and Methods

2.3.1. Cells

Human embryonic kidney 293T cells were cultured in 5% CO 2 in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal calf serum (Gibco) at 37ºC.

2.3.2. Plasmids

The UL76 gene from HCMV and the ORF20 homologue from MHV- 68 were cloned into pcDNA3.1 plasmid fused in frame with an amino-terminal influenza haemaglutinin peptide (HA) tag. The luciferase reporter construct containing human IL-8 promoter was a gift from Dr Naofumi Mukaida and has been described before (Murayama et al. , 1997). The pCMV β plasmid contains a β- galactosidase gene under the control of human cytomegalovirus immediate early promoter, and serves as an internal control for variations in transfection efficiency. Lentivirus vectors pHR-CMV-

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Chapter 2 eGFP, the envelope HCMV-VSVG and packaging pCMVR8.9 were described previously (Ikeda et al. , 2002). The UL76 and ORF20 gene fused with HA tag were excised from pcDNA3.1 plasmid by BamHI/XhoI digestion and cloned into the pHR-CMV-eGFP vector (pHR-CMV-eGFP-UL76 and pHR-CMV-eGFP-ORF20, respectively).

2.3.3. Lentivirus production and titration

Lentiviruses were produced by transient transfection of 293T cells with a weight ratio of 3:1:1 of vector to packaging to envelope plasmids using Fugene 6 (Roche) according to the manufacturer’s instructions. Control lentivirus was produced by co-transfection of the packaging and envelope plasmid simultaneously with the empty pHR-CMV-eGFP plasmid. For production of recombinant lentiviruses expressing UL76 or ORF20, the plasmids pHR-CMV- eGFP-UL76 or pHR-CMV-eGFP-ORF20 were used. Supernatants of transfected cells were collected at 48h, 72h, and 96h post- transfection and, after a pre-clearing by low speed centrifugation, the lentiviruses were collected by ultracentrifugation at 25000 rpm for 3h at 4ºC. Lentiviruses pellets were resuspended in fresh culture medium and frozen at -80ºC. Lentivirus titers were determined by infection of 293T cells with a dilution factor of 4, followed by detection of eGFP positive cells through flow cytometry at 48h post-infection.

2.3.4. Lentivirus transduction and RNA isolation

293T cells were infected with control or recombinant lentiviruses expressing UL76 or ORF20 for one hour with shaking. Transduced cells were cultured in fresh medium for 48h before harvesting of cells and RNA isolation. Total RNA of cells transduced with control,

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UL76 or ORF20 recombinant lentiviruses was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Concentration and purity of the RNA was determined by spectrophotometry and its integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA).

2.3.5. Target Synthesis and Hybridization to Affymetrix GeneChips

RNA was processed for use on Affymetrix GeneChip Human Genome U133A 2.0 Arrays (Santa Clara, CA, USA), according to the manufacturer’s protocol. Briefly, 5 µg of total RNA containing added Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (One-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription reaction to generate biotinylated cRNA (GeneChip Expression 3’-Amplification Reagents for IVT-Labeling; Affymetrix). Size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay. Ten micrograms of fragmented cRNA was mixed in a 200µl hybridization reaction containing hybridization controls, followed by hybridization on arrays for 16h at 45°C. Standard post- hybridization wash and double-stain protocols were used on an Affymetrix GeneChip Fluidics Station 400 and arrays were scanned on an Affymetrix GeneChip scanner 3000. All quality parameters for the arrays were confirmed to be in the recommended range.

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2.3.6. Microarrays Data Analysis

Scanned arrays were initially analyzed with Affymetrix MAS 5.0 software to obtain Absent/Present calls and with dChip 2005 for subsequent analysis. The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method (Li & Wong, 2001). Normalized CEL intensities of the arrays were used to obtain model-based gene expression indices based on a PM (Perfect Match)-only model (Li & Wong, 2001). Replicate data for the same sample type were weighted gene-wise by using inverse squared standard error as weights. All genes compared were considered to be differentially expressed if the 90% lower confidence bound of the fold change between experiment and baseline was above 1.5. The lower confidence bound criterion means that we can be 90% confident that the fold change is a value between the lower confidence bound and a variable upper confidence bound. Annotations for the >18.400 transcripts that are represented on the GeneChip Human Genome U133A 2.0 Arrays were obtained from the NetAffx database (www.affymetrix.com) as of December 2005 and imported into dChip using ChipInfo software (Zhong et al. , 2003).

2.3.7. Reverse transcriptase polymerase chain reaction (RT- PCR)

The RNA from 293T cells transduced with control, UL76 or ORF20 recombinant lentiviruses, or from cells stimulated with 10 ng/ml TNF α for 5h (positive control), were treated with DNase I (Invitrogen) at room temperature for 15 minutes and then incubated at 65°C for 10 minutes. First strand cDNA was synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV

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RT) (Invitrogen) and incubated at 37°C for 50 minutes followed by incubation at 70ºC for 15 minutes to inactivate the reaction. For reverse transcriptase PCR, the following specific primers for IL-8 were used: IL-8F 5’- ATGACTTCCAAGCTGGCCGTGGCTCTCTTGG-3’; IL-8R 5’- GATAAATTTGGGGTG GAAAGG-3’. Samples were amplified for 25 cycles at 94°C for 1 minute, at 55°C for 1 minute and at 72°C for 2 minutes. The PCR products were electrophoresed through a 3% agarose gel and visualized with ethidium bromide.

2.3.8. Luciferase assays

293T cells were co-transfected in triplicate with 100 ng of IL-8 luciferase reporter plasmid, 25 ng of the β-galactosidase internal control plasmid (pCMV β) and with the indicated amounts of either pcDNA3.1HA-UL76 or pcDNA3.1HA, according to the Lipofectamine 2000 (Invitrogen) protocol. Cells were lysed 28h-30h post-transfection and the luciferase activity was measured using the luciferase assay system (Promega) according to the manufacturer`s protocol. β-galactosidase activity was measured using the Galacton-Plus kit from Tropix (Bedford, MA). The luciferase activity was normalized relative to the β-galactosidase activity of each sample as control of transfection efficiency.

2.3.9. Enzyme-linked Immunoabsorbent Assay (ELISA)

Supernatants of 293T cells transfected with pcDNA3.1 (negative control) or pcDNA3.1HA-UL76 plasmids according to the Lipofectamine 2000 (Invitrogen) protocol were collected at 48h post-transfection. The concentration of secreted IL-8 was determined following the manufacturer’s instructions (IL-8 ELISA kit, BD Biosciences). Plates were analyzed at 450 nm using a

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BioRad ELISA Reader (BioRad) and concentration of IL-8 was determined by comparison to a standard curve.

2.3.10. Western blot

Total lysates of transfected 293T cells used in luciferase assays or ELISA were loaded on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). The separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare) and blocked with 5% nonfat milk for one hour at room temperature. Membranes were incubated with an anti-HA (Roche) and anti-β-actin (Sigma) horseradish peroxidase-conjugated antibodies. Immunoblots were developed by enhanced chemiluminescence detection according to the manufacturer's instructions (ECL, Amersham Pharmacia Biotech).

2.3.11. Statistical Analysis

Data were shown as mean values with standard deviation (SD). Differences between experimental groups were determined by a two-tailed Student t test using GraphPad Prism 5 software.

2.4. Results

2.4.1. The HCMV UL76 gene induces transcription of IL-8

In order to elucidate the impact of UL24 homologues on intracellular signaling pathways we used DNA microarrays in combination with bioinformatics. This technology enables the simultaneous detection of genes that are active or inactive in different samples and the different levels of expression between samples. Transcription profiles of 293T cells expressing UL24 homologues from MHV-68 (ORF20) or HCMV (UL76) were

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Chapter 2 analyzed and compared to control transduced cells. The complete list of up-regulated and down -regulated genes is presented as supplemental data in the Annex section. As expected, expression of both genes resulted in increased expression of several genes associated with cell cycle and apoptosis pathways. Othe r cellular genes, however, were only affected by one of the UL24 homologues. One of these genes, whose expression was up - regulated (fold change 4,94) in cells expressing UL76, but not in ORF20, was interleukin-8. To confirm the results from microarray anal ysis, RNA was extracted from cells transduced with lentivirus es expressing ORF20, UL76 or control and cDNA was prepared for each sample. RNA extracted from 293T cells stimulated with TNF α served as a positive control. Semi-quantitative reverse transcriptase PCR was performed using specific primers for IL -8 and tubulin as internal control. Consistent with microarray analysis, expression of UL76, but not ORF20, increases the level of IL -8 mRNA compared to the control plasmid (Fig.2.1).

Figure 2.1. UL76 induces IL -8 gene transcription. Total RNAs of cells transduced with control, ORF20 or UL76 -expressing lentivirus were subjected to RT-PCR using primers specific for IL -8 (top) and tubulin as an internal control (bottom). As positive control, RNA was extracted from 293T cells stimulated with TNF α (10 ng/ml).

Interestingly, it is known that induction of IL -8 is a critical event during HCMV infection as it impacts positively on viral replication

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Chapter 2 and virion production (Craigen et al. , 1997; Murayama et al. , 1997). Thus, the ability of UL76, the UL24 homologue from HCMV, to induce IL-8 expression was studied in more detail. The effect of UL76 expression on the transcription activation of IL-8 was evaluated using a luciferase reporter construct containing the IL-8 promoter sequence (Murayama et al. , 1997). Luciferase activity was determined in lysates of cells co-transfected with the IL-8 luciferase reporter construct and UL76 expression plasmid or control vector. As shown in Fig.2.2, expression of UL76 significantly activated transcription of IL-8 promoter. This effect was specific and dose-dependent as increasing amounts of UL76 resulted in higher activation of IL-8 promoter (Fig.2.2).

Figure 2.2. UL76 activates IL-8 promoter transcription. 293T cells were co-transfected with pCDNA3.1 or pCDNA3.1HA-UL76, IL-8 luciferase reporter and β-Galactosidase plasmid. Luciferase activity was measured in cell lysates at 28h-30h post-transfection. Luciferase activity was normalized to β-Galactosidase activity as a control for transfection efficiency. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Statistical significance is represented as ** p<0.01 ; *** p<0.001 .

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2.4.2. HCMV UL76 gene induces secretion of IL-8 protein

To examine the IL-8 induction at the level of protein expression, we determined the amount of IL-8 secreted into the supernatants of cells expressing UL76 or control plasmid by ELISA. As shown in Figure 2.3, cells expressing UL76 secreted significantly higher levels of IL-8 as compared to the control vector ( p<0.01 ). These results are consistent with the IL-8 up-regulation observed previously at the level of gene transcription. In conclusion, UL76 induces IL-8 expression both at the level of transcriptional activation and protein secretion.

Figure 2.3. UL76 induces IL-8 secretion. 293T cells were transfected with 300 ng of control plasmid or UL76 expression plasmid. Supernatants were collected 48h post-transfection and IL-8 concentration (pg/ml) was determined by ELISA. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. ** Statistically significant as compared with control vector-expressing cells (p<0.01) . Expression of UL76 HA-tagged was confirmed by western blot (below) using an anti-HA HRP-conjugated antibody and β-actin detection was used as loading control.

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2.5. Discussion

Conserved herpesviruses protein families are one promising approach to explore new viral targets for the development of a global anti-herpesvirus therapy or protective vaccines. The UL24 gene family is conserved in all herpesviruses but does not have any sequence homology to cellular genes, thus their viral function can only be accessed by functional or structural assays. Using this approach, our group demonstrated that UL24 human and murine homologues induce cell cycle arrest (Nascimento et al. , 2009; Nascimento & Parkhouse, 2007). The precise mechanism of cell cycle arrest induced by UL24, however, remains to be clarified. In order to indentify intracellular signaling pathways that are modulated by UL24, DNA microarrays analysis was performed in cells expressing UL24 homologues from MHV-68 (ORF20) and from HCMV (UL76). Comparison of the cellular transcription profiles revealed an up-regulation of several proteins involved in the cell cycle and in apoptosis (see Anex), as expected from our previous results. Other genes, however, were only affected by one of the UL24 homologues and may be related to the specific life cycle of the virus or, since MHV-68 is a γ-herpesvirus and HCMV is a β-herpesvirus, they may be related to different characteristics of these two herpesvirus subfamilies. This work focused on one of these genes, IL-8, whose expression was up-regulated in cells expressing UL76, but not by ORF20, the UL24 homologues from HCMV and MHV-68 respectively. Interleukin-8, a pro-inflammatory CXC chemokine, is induced by a wide range of extracellular stimuli, including virus, and attracts neutrophils and lymphocytes (Baggiolini & Clark-Lewis, 1992).

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Here, it was demonstrated that expression of UL76 not only resulted in transcriptional activation of the IL-8 promoter, but also stimulated increased levels of secreted IL-8 protein. Thus, UL76 must contribute to the induction of IL-8 previously described for HCMV infection (Murayama et al. , 1997). Recruitment of neutrophils to the site of infection mediated by IL-8 may be critical for HCMV propagation, as neutrophils can acquire infectious virus from infected endothelial cells and disseminate the virus via the bloodstream (Craigen et al. , 1997). In addition, IL-8 has a positive impact on HCMV replication (Murayama et al. , 1994). Thus, it is important to identify the genes involved in IL-8 induction by HCMV as they may be novel targets for the control of HCMV infection and its pathogenesis. The failure of ORF20 to up-regulate IL-8 expression may be related to the fact that there is no murine IL-8 homologue. Nevertheless, there are functional homologues, such as KC or MIP-2, which are also CXC chemokines and primarily chemoattractants for neutrophils (Bozic et al. , 1995; Driscoll et al. , 1995). The impact of ORF20 on the expression of these murine chemokines, however, was not evaluated as the DNA microarray analysis was performed with human 293T cells. Immediately relevant, however, is the fact that increased levels of KC and MIP-2 were detected in mice infected with MHV-68 (Sarawar et al. , 2002) and KC was shown to enhance MHV-68 replication in permissive fibroblasts (Lee et al. , 2003). Therefore, the impact of OR20 in the expression of KC or MIP-2 in mice should be explored in the future. In conclusion, in this chapter, UL76 was identified as an IL-8- inducing HCMV viral gene. The characterization of the mechanism of IL-8 induction by UL76 and its impact in the context of viral infection will be the aim of the next chapters.

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2.6. References

Aihara, M., Tsuchimoto, D., Takizawa, H., Azuma, A., Wakebe, H., Ohmoto, Y., Imagawa, K., Kikuchi, M., Mukaida, N. & Matsushima, K. (1997). Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45. Infect Immun 65 , 3218-3224. Baggiolini, M. & Clark-Lewis, I. (1992). Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett 307 , 97-101. Bozic, C. R., Kolakowski, L. F., Gerard, N. P., Garcia-Rodriguez, C., von Uexkull-Guldenband, C., Conklyn, M. J., Breslow, R., Showell, H. J. & Gerard, C. (1995). Expression and biologic characterization of the murine chemokine KC. J Immunol 154 , 6048-6057. Cooray, S., Bahar, M. W., Abrescia, N. G., McVey, C. E., Bartlett, N. W., Chen, R. A., Stuart, D. I., Grimes, J. M. & Smith, G. L. (2007). Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. J Gen Virol 88 , 1656-1666. Cota, M., Kleinschmidt, A., Ceccherini-Silberstein, F., Aloisi, F., Mengozzi, M., Mantovani, A., Brack-Werner, R. & Poli, G. (2000). Upregulated expression of interleukin-8, RANTES and chemokine receptors in human astrocytic cells infected with HIV- 1. J Neurovirol 6, 75-83. Craigen, J. L., Yong, K. L., Jordan, N. J., MacCormac, L. P., Westwick, J., Akbar, A. N. & Grundy, J. E. (1997). Human cytomegalovirus infection up-regulates interleukin-8 gene expression and stimulates neutrophil transendothelial migration. Immunology 92 , 138-145. Davison, A. J., Dargan, D. J. & Stow, N. D. (2002). Fundamental and accessory systems in herpesviruses. Antiviral Res 56 , 1-11. DeForge, L. E., Preston, A. M., Takeuchi, E., Kenney, J., Boxer, L. A. & Remick, D. G. (1993). Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem 268 , 25568-25576. Driscoll, K. E., Hassenbein, D. G., Howard, B. W., Isfort, R. J., Cody, D., Tindal, M. H., Suchanek, M. & Carter, J. M. (1995). Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol 58 , 359-364. Hoheisel, J. D. (2006). Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet 7, 200-210. Holzerlandt, R., Orengo, C., Kellam, P. & Albà, M. M. (2002). Identification of new herpesvirus gene homologs in the human genome. Genome Res 12 , 1739-1748. Ikeda, Y., Collins, M. K., Radcliffe, P. A., Mitrophanous, K. A. & Takeuchi, Y. (2002). Gene transduction efficiency in cells of different species by HIV and EIAV vectors. Gene Ther 9, 932-938.

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Kasahara, T., Mukaida, N., Yamashita, K., Yagisawa, H., Akahoshi, T. & Matsushima, K. (1991). IL-1 and TNF-alpha induction of IL-8 and monocyte chemotactic and activating factor (MCAF) mRNA expression in a human astrocytoma cell line. Immunology 74 , 60- 67. Lee, B. J., Koszinowski, U. H., Sarawar, S. R. & Adler, H. (2003). A gammaherpesvirus G protein-coupled receptor homologue is required for increased viral replication in response to chemokines and efficient reactivation from latency. J Immunol 170 , 243-251. Li, C. & Wong, W. H. (2001). Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci U S A 98 , 31-36. Medin, C. L. & Rothman, A. L. (2006). Cell type-specific mechanisms of interleukin-8 induction by dengue virus and differential response to drug treatment. J Infect Dis 193 , 1070-1077. Murayama, T., Kuno, K., Jisaki, F., Obuchi, M., Sakamuro, D., Furukawa, T., Mukaida, N. & Matsushima, K. (1994). Enhancement human cytomegalovirus replication in a human lung fibroblast cell line by interleukin-8. J Virol 68 , 7582-7585. Murayama, T., Ohara, Y., Obuchi, M., Khabar, K. S., Higashi, H., Mukaida, N. & Matsushima, K. (1997). Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB- binding sites of the interleukin-8 gene. J Virol 71 , 5692-5695. Nascimento, R., Dias, J. D. & Parkhouse, R. M. (2009). The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex. Arch Virol 154 , 1143-1149. Nascimento, R. & Parkhouse, R. M. (2007). Murine gammaherpesvirus 68 ORF20 induces cell-cycle arrest in G2 by inhibiting the Cdc2- cyclin B complex. J Gen Virol 88 , 1446-1453. Pellett, P. E. & Roizman, B. (2007). The Family Herpesviridae: A Brief Introduction. In Fields Virology, 5th Edition . Edited by D. M. Knipe & P. M. Howley: Lippincott Williams & Wilkins. Sarawar, S. R., Lee, B. J., Anderson, M., Teng, Y. C., Zuberi, R. & Von Gesjen, S. (2002). Chemokine induction and leukocyte trafficking to the lungs during murine gammaherpesvirus 68 (MHV-68) infection. Virology 293 , 54-62. Siew, V. K., Duh, C. Y. & Wang, S. K. (2009). Human cytomegalovirus UL76 induces chromosome aberrations. J Biomed Sci 16 , 107. Zhong, S., Li, C. & Wong, W. H. (2003). ChipInfo: Software for extracting gene annotation and gene ontology information for microarray analysis. Nucleic Acids Res 31 , 3483-3486.

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CHAPTER 3

MECHANISM OF IL-8 INDUCTION BY HCMV UL76 PROTEIN

Chapter 3

3.1. Summary

In this chapter, the mechanism of IL-8 induction by UL76, the UL24 homologue of HCMV was explored. Deletion mutant analysis of the IL-8 promoter using luciferase assays indicated that induction of IL- 8 by UL76 is dependent on the NF-kB transcription factor, although AP-1 or NF-IL-6 may also contribute to optimal IL-8 induction. Activation of NF-kB pathway by UL76 was then confirmed by luciferase assay using a luciferase reporter construct responsive to NF-kB. The observations that induction of IL-8 by UL76 requires functional IKK β, the degradation of the IkB protein and promotes the translocation of p65(RelA) subunit to the nucleus, indicate that UL76 activates the NF-kB canonical pathway. Activation of this pathway can result from diverse stimuli, generally involving membrane-associated receptor stimulation. A different mechanism, however, has been recently studied and is initiated from genotoxic stress. The fact that UL76 localizes in the nucleus together with the observation that UL76 activates DNA damage suggested that this pathway could be involved in the activation of NF-kB by UL76 (Siew et al. , 2009). Indeed, and similar to the impact of genotoxic drugs, expression of UL76 resulted in a nuclear accumulation and phosphorylation at serine 85 of NEMO. Finally, inhibition of ATM using a specific ATM inhibitor or an ATM knockout cell line resulted in abrogation of IL-8 induction by UL76. Collectively, these results demonstrate that IL-8 induction by UL76 is NF-kB and ATM- dependent. The ability of UL76 to regulate IL-8 expression and the cell cycle is not due to its putative endonuclease activity, although the level of IL-8 induction was reduced when the putative endonuclease motifs were mutated.

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3.2. Introduction

Interleukin-8, a prototypic human chemokine, is produced by most normal and tumor cells. The expression of IL-8 mRNA and the release of the biologically active protein have been observed in many different cell types, such as endothelial cells, fibroblasts from different tissues, keratinocytes, synovial cells, chondrocytes, epithelial cells, several tumor cells and even neutrophils (Baggiolini & Clark-Lewis, 1992). Although expression of IL-8 is low or absent under normal conditions, it is highly inducible by a wide range of extracellular stimuli such as pro-inflammatory cytokines (e.g. IL-1, TNF α) (Kasahara et al. , 1991), bacterial (Aihara et al. , 1997) or viral infections (Cota et al. , 2000; Medin & Rothman, 2006). Excessive amounts of locally produced IL-8 can have deleterious effects. Accordingly, IL-8 gene expression is tightly controlled at both transcriptional and post-transcriptional levels. The IL-8 promoter region contains binding sites for the NF-kB, AP- 1 and NF-IL-6 transcription factors. Although the NF-kB site is required for induction of IL-8 in the majority of cell types studied, the contribution of the AP-1 and NF-IL-6 sites to optimal IL-8 activation may vary with the stimulus and the cell type (Hoffmann et al. , 2002). The NF-kB canonical, or “classical”, pathway involves the activation of the IKK complex, consisting of two catalytic kinase subunits, IKK α and IKK β, and a regulatory subunit, IKK γ/NEMO. In most unstimulated cells, NF-kB heterodimers (mostly p65/p50 dimers) localize in the cytoplasm in the inactive form as a result of their interaction with the IkB proteins. Upon stimulation, IkB is phosphorylated by the IKK complex, ubiquitinated and targeted for degradation, thus releasing the NF-kB subunits which translocate to the nucleus and induce transcription of target genes. The non-

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Chapter 3 canonical pathway, on the other hand, includes activation of NF- kB-inducing kinase (NIK) and subsequent activation of IKK α dimer. Phosphorylation of the p100 NF-kB subunit by IKK α induces its proteolytic processing to p52 and results in the activation of heterodimers containing this subunit and, in most cases, RelB (Perkins, 2007). Although most of the physiological inducers of NF-kB involve the canonical and non-canonical pathways, in recent years alternative mechanisms leading to NF-kB nuclear localization and DNA binding have been identified. One of these pathways is induced by DNA damage activation and, in contrast to inflammatory stimuli such as TNF α or IL-1β, the signal originates in the nucleus (Wu et al. , 2006). Activation of NF-kB by genotoxic stress requires induction of two independent parallel pathways. The first one leads to SUMOylation of NEMO through a mechanism dependent on PARP1, PIASy and Ubc9. A second pathway triggered upon DNA damage results in the phosphorylation and activation of the kinase ATM. Activation of both these pathways leads to phosphorylation and ubiquitination of sumoylated NEMO in an ATM-dependent way. Ubiquitinated NEMO associated with ATM is exported back to the cytoplasm, activating the IKK complex and subsequent NF-kB activation, in a similar manner to the canonical pathway described above (Miyamoto, 2011). In this chapter, the mechanism of IL-8 induction by UL76 is characterized in detail. Deletion mutant analysis of IL-8 promoter showed that UL76 induces this chemokine essentially through the NF-kB pathway. Further analysis demonstrated that UL76 is able to activate NF-kB as result of DNA damage pathway induction, a finding consistent with the nuclear localization of the UL76 protein.

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3.3. Materials and Methods

3.3.1. Cells

Human embryonic kidney 293T cells were cultured in 5% CO 2 in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal calf serum (Gibco) at 37ºC. Human foreskin fibroblasts (HFF) (obtained from European Collection of Cell Cultures) and A- T human fibroblast cell line (ATM -/- GM09607) (obtained from the Coriell Institute for Medical Research) were cultured in Minimum Essential Medium with Earle's salts (Gibco) supplemented with 10% fetal calf serum (Gibco).

3.3.2. Plasmids

The UL76 gene from HCMV was cloned into pcDNA3.1 plasmid fused in frame with an amino-terminal influenza haemaglutinin peptide (HA) tag. The three putative endonuclease amino acids in the UL76 gene were mutated to glycine (pcDNA3.1HA-E/K mut plasmid) according to the Directed Mutagenesis kit protocol (Stratagene). The luciferase reporter constructs containing human IL-8 promoter (-131) or a mutation in NF-kB, AP-1 or NF-IL-6 binding site were a gift from Dr Naofumi Mukaida and have been described before (Murayama et al. , 1997). The reporter plasmid for NF-κB [p(PRD2)5tk ∆(-39)lucter] was a gift from Dr Steve Goodbourn. Dominant negative mutants of IKK β and IkB α (S32/36A) plasmids including an HA tag, were obtained from Dr Michael Karin (Zandi et al. , 1998) and Dr Dean Ballard (Brockman et al. , 1995), respectively. The pCMV β plasmid contains a β- galactosidase gene under the control of human cytomegalovirus immediate early promoter, and serves as an internal control for variations in transfection efficiency.

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3.3.3. Luciferase assays

293T cells were co-transfected in triplicate with 100 ng of IL-8 luciferase reporter plasmid or luciferase reporter constructs containing mutations in the IL-8 promoter ( ∆NF-kB, ∆AP-1 and ∆NF-IL-6), 25 ng of β-galactosidase internal control plasmid (pCMV β) and 300 ng of pcDNA3.1 or pcDNA3.1HA-UL76, according to the Lipofectamine 2000 (Invitrogen) protocol. A similar transfection protocol was performed using the NF-kB luciferase reporter plasmid. Cells were lysed 28h-30h post-transfection and the luciferase activity was measured using the luciferase assay system (Promega) according to the manufacturer`s protocol. β- galactosidase activity was measured using the Galacton-Plus kit from Tropix (Bedford, MA). The luciferase activity was normalized relative to the β-galactosidase activity of each sample as control of transfection efficiency.

3.3.4. Immunofluorescence

The 293T or HFF cells were cultured on sterile glass coverslips and transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid according to the Lipofectamine 2000 or LTX (Invitrogen) protocol. As positive control cells were stimulated with recombinant human TNF α (10 ng/ml) (Peprotech) for 30 minutes. At the indicated times post-transfection, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 minutes. Fixed cells were permeabilised with PBS-0.1% Triton X-100 for 20 minutes. After washing, the cells were blocked with PBS-0.05% Tween 20 containing 5% normal goat serum for one hour. The samples were incubated with anti-p65 (F-6) or anti-IKK γ/NEMO (B-3) (Santa Cruz Biotechnology, Inc) followed by incubation with anti-mouse Texas Red secondary

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Chapter 3 antibody (Molecular Probes) and anti-HA-FITC conjugated antibody (Roche) to visualize UL76 HA-tagged protein. After incubation with DAPI, the coverslips were mounted on “Slow Fade” (Invitrogen) and examined under a fluorescence microscope.

3.3.5. Nuclear extracts preparation

293T cells were transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid according to the Lipofectamine 2000 (Invitrogen) protocol. Nuclear extraction was performed using a Nuclear Extraction Kit according to the manufacturer’s indications (Active Motif). As positive control, cells were stimulated for 2h with 10 µM of etoposide (Sigma). Briefly, at the indicated times post- transfection, cells were collected in ice-cold PBS in the presence of phosphatase inhibitors. Cytoplasmic extracts were obtained by resuspending the cells in hypotonic buffer followed by addition of detergent. After centrifugation the pelleted nuclei was solubilized in the lysis buffer supplemented with a protease inhibitor cocktail. Protein concentrations were determined by Bradford assay (Bio- Rad Laboratories).

3.3.6. Western blot

Total lysates from cells transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid were prepared using lysis buffer supplemented with a mixture of protease and phosphatase inhibitors (Calbiochem), for 30 minutes on ice. Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories). For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), total or nuclear lysates were loaded per lane and the separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare) and

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Chapter 3 blocked with 5% nonfat milk for one hour at room temperature. Membranes were incubated with a monoclonal antibody for p65 (F- 6) or IKK γ/NEMO (B-3), nucleolin/C23 (H-250) (Santa Cruz Biotechnology, Inc), IKK γ/NEMO(S85) (Assay Biotech), tubulin, anti-HA and anti-β-actin horseradish peroxidase-conjugated antibody (Sigma). Horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma. Immunoblots were developed by enhanced chemiluminescence detection according to the manufacturer's instructions (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).

3.3.7. Enzyme-Linked Immunoabsorbent Assay (ELISA)

Supernatants of 293T or ATM -/- cells transfected with pcDNA3.1 (negative control) or pcDNA3.1HA-UL76 plasmids according to the Lipofectamine 2000 (Invitrogen) protocol were collected at 48h post-transfection. As control, cells were stimulated for 5h with etoposide (Sigma) at a dose of 10 µM or TNF α (20 ng/ml). The concentration of IL-8 secreted was determined using an IL-8 ELISA kit (BD Biosciences) and following the manufacturer’s instructions. Plates were analyzed at 450 nm using a BioRad ELISA Reader (BioRad) and levels of IL-8 were determined by comparison to a standard curve.

3.3.8. Cell cycle analysis

293T cells were infected with recombinant pHR-CMV-eGFP-UL76, pHR-CMV-E/K mut or control lentivirus. Cells were collected by trypsinizing (Gibco BRL) 48h post-transfection, washed once with PBS and fixed with 90% ethanol overnight at 4ºC. After fixation, cells were washed with PBS, resuspended in PBS-0.01% Triton-X

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100 and incubated with 50U DNAse-free RNAse A (Calbiochem) for 30 minutes at room temperature. After incubation, cells were stained with propidium iodide (Sigma). Flow cytometry analysis was performed using FACS Calibur instrument (Becton Dickinson) and cell cycle analysis was performed using CellQuest software.

3.3.9. Statistical analysis

Data were shown as mean values with standard deviation (SD). Differences between experimental groups were determined by a two-tailed Student t test using GraphPad Prism 5 software.

3.4. Results

3.4.1. Induction of IL-8 by HCMV UL76 is NF-kB-dependent

Expression of IL-8 is tightly regulated at the transcription level. The sequence of nucleotides -1 to -133 in the promoter region of IL-8 gene is essential for its transcription regulation and contains binding sites for NF-kB, AP-1 and NF-IL-6 transcription factors (Fig.3.1A) (Hoffmann et al. , 2002). To determine the mechanism of IL-8 induction by UL76, we compared the luciferase activity of wild type IL-8 luciferase reporter with its mutant derivatives containing a mutation in each transcription factor binding site in cells expressing UL76 or control plasmid. There was no significant difference in luciferase activity when AP-1 or NF-IL-6 binding sites were mutated in the luciferase reporter construct as compared to wild-type IL-8 promoter. The effect of UL76 in IL-8 transcriptional activation, however, was drastically reduced in the absence of the NF-kB binding site, suggesting a critical role for the NF-kB pathway in the UL76 mediated induction of IL-8 (Fig.3.1B).

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The hypothesis that UL76 up-regulates IL-8 expression by activating the NF-kB pathway was tested using a luciferase reporter construct responsive to NF-kB and measuring the luciferase activity in cells expressing UL76 or control vector. Consistent with previous results, UL76 significantly activated the NF-kB luciferase reporter when compared to the control plasmid (Fig.3.1C).

Figure 3.1 IL-8 induction by HCMV UL76 is NF-kB-dependent. A) Schematic representation of 5'-flanking region of human IL-8 promoter from -131 to +1 base pair, demonstrating locations of defined binding sites for NF-KB, NF-IL-6 and AP-1. The arrow downstream of the TATA box

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Chapter 3 indicates the start of transcription (+1). B) Luciferase activity was measured in 293T cells co-transfected with pcDNA3.1 or pcDNA3.1HA- UL76 and wild-type IL-8 promoter or each one of the deletion mutants luciferase reporter constructs. Luciferase activity was normalized to β- Galactosidase activity as control of transfection efficiency. Data is expressed as fold induction of control vector and is representative of three similar experiments. The x indicates the mutation on AP-1, NF-IL-6 and NF-kβ binding sites within the IL-8 promoter. Statistical significance compared to IL-8 wild type promoter is represented as * p<0.05 C) UL76 activates NF-kB pathway. Luciferase activity was determined in 293T cells co-transfected with pCDNA3.1 or pCDNA3.1HA-UL76 and the NF-kB luciferase reporter as previously described. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Statistical significance is represented as ** p<0.01.

3.4.2. Induction of IL-8 by UL76 requires IKK β activation and IkB α degradation.

To further characterize the NF-kB pathway activation by UL76, we used a catalytically inactive mutant IKK β and a phosphorylation- resistant mutant IkB α (IkB αS32/36A), in which the two critical serine residues were mutated to alanine and, consequently, blocking its phosphorylation and degradation. Both constructs function as dominant negatives, inhibiting the activity of cellular wild type IKK β and IkB α, respectively. Co-transfection of each dominant negative with IL-8 luciferase reporter and UL76 expression plasmid or control vector resulted in a reduction of IL-8 induction in cells expressing UL76 (Fig.3.2). These results confirm the critical role of the NF-kB pathway in IL-8 induction by UL76.

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Figure 3.2 Induction of IL-8 by UL76 requires IKK β activation and IkB α degradation. Luciferase activity was determined in 293T cells co- transfected with pcDNA3.1 or pcDNA3.1HA-UL76, IL-8 luciferase reporter and dominant negative IKK β or IkB α (S32/36A) plasmids. Values of luciferase activity were normalized to β-Galactosidase activity. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Expression of UL76 and dominant negatives IKK β or IkB α (S32/36A) was confirmed by western blot (below) using an anti-HA HRP- conjugated antibody. Tubulin was used as loading control.

3.4.3. UL76 induces translocation of p65 to the nucleus

After IkB α degradation by the proteosome, heterodimers of NF-kB subunits translocate to the nucleus where they bind to the target gene promoter region and activate transcription (Perkins, 2007). Thus, we evaluated the effects of UL76 expression on the subcellular localization of NF-kB p65 subunit by immunofluorescence analysis using an anti-p65 antibody. The cells were co-stained with an anti-HA-FITC conjugated antibody to

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Chapter 3 confirm expression of the UL76-HA tagged protein. As shown in Figure 3.3A, p65 was mainly localized in the nucleus of the UL76- transfected HFF cells, in contrast to its cytoplasm localization in control cells (Fig. 3.3A). Similar results were obtained in 293T nuclear extracts (Fig. 3.3B). Thus, immunoblotting with anti-p65 antibody revealed an accumulation of p65 in the nucleus of cells expressing UL76. This accumulation was specific, as can be seen from the constant levels of the nucleolin expression in the loading control. As positive control, cells were stimulated with etoposide, a genotoxic stress drug that activates NF-kB pathway in response to DNA damage (Fig. 3.3B).

Collectively, these results indicate that IL-8 induction by UL76 results from activation of the NF-kB pathway. Specifically, it was demonstrated that UL76 activates IKK β and consequent degradation of IkB α to promote the translocation of the p65 subunit to the nucleus, where it may bind to IL-8 promoter.

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Figure 3.3 UL76 induces p65 translocation to the nucleus. A) Indirect immunofluorescence was performed on HFF cells transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid using an anti-p65 antibody and anti-mouse Texas Red conjugated secondary antibody. UL76 expression was detected using HA-FITC conjugated antibody and DAPI staining was used to define nuclei. B) 293T cells transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid were lysed for nuclear extraction at the indicated time points post-transfection. Nuclear extracts were immunobloted with anti-p65 and anti-nucleolin antibody as loading control. Expression of UL76 was detected using an anti-HA-HRP conjugated antibody.

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3.4.4. IL-8 induction by HCMV UL76 is ATM-dependent In agreement with our own unpublished results, a recent report indicated that UL76 induces DNA damage (Siew et al. , 2009). Interestingly, several studies have been characterizing an alternative pathway to NF-kB activation that result from DNA damage. Based on this, we hypothesized that the ability of UL76 to induce DNA Damage resulted both in cell cycle arrest and induction of IL-8 expression through activation of NF-kB pathway. A characteristic feature of NF-kB pathway activation by genotoxic stress is the accumulation of IKK γ/NEMO in the nucleus where a series of post-translational modifications occurs (Wu et al. , 2006). Immunostaining using an anti-IKK γ/NEMO antibody revealed increasing amounts of nuclear IKK γ/NEMO in cells expressing the UL76-HA tagged protein (Fig. 3.4A). The nuclear post-translational modifications of NEMO that are critical for NF-κB activation following genotoxic stress include ATM-independent sumoylation and ATM-dependent phosphorylation at serine 85 followed by monoubiquitination. Immunoblotting of 293T cell lysates with a specific IKK γ/NEMO(S85) antibody demonstrated that expression of UL76 induces phosphorylation of IKK γ/NEMO as observed upon genotoxic stress. These results indicate that ATM has a critical role in the activation of NF-kB pathway by UL76 (Fig. 3.4B).

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Figure 3.4. UL76 activates NEMO through DNA damage pathway. A) UL76 expression leads to IKK γ/NEMO NEMO nuclear accumulation. Immunofluorescence was performed on 293T cells transfected with pcDNA3.1HA-UL76 or control pcDNA3.1 plasmid using an anti- IKK γ/NEMO antibody. UL76 expression was detected using HA-FITC conjugated antibody and DAPI staining was used to define nuclei. (B) UL76 expression induces phosphorylation of NEMO at serine 85. 293T cells transfected with pcDNA3.1HA-UL76 or control plasmid were lysed at 24h post-transfection and total lysates were analyzed by western blot. Expression of UL76 was confirmed using an anti-HA-HPR conjugated antibody and tubulin was used as loading control.

To evaluate the impact of ATM in IL-8 induction by UL76, we used two different approaches: a specific ATM inhibitor, KU55933, and a human fibroblast cell line deficient in ATM. Supernatants of 293T

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Chapter 3 cells expressing UL76, or the control plasmid, in the presence or absence of KU55933, were collected at 48h post-transfection and the IL-8 concentration was determined by ELISA. Inhibition of ATM by KU55933 blocked the UL76-induced IL-8 secretion (Fig. 3.5A). Similarly, IL-8 concentration was determined in supernatants of ATM -/- cells expressing UL76 or the control plasmid. As shown in Figure 3.5B, UL76 or etoposide, a genotoxic drug, are unable to induce IL-8 in the absence of ATM. Although UL76 expression leads to higher levels of IL-8 than etoposide stimulation, there is no increase in IL-8 secretion when compared to control vector, so this basal induction is possibly due to transfection. Moreover, this result is not due to the incapacity of this cell line to produce IL-8 since stimulation with TNF α, a membrane receptor-triggering NF-kB canonical pathway independent of ATM, is still capable of inducing IL-8 secretion (Fig. 3.5B). Expression of UL76 was confirmed in cell lysates by western blot. In summary, these results indicate that activation of NF-kB pathway and consequent IL-8 induction by UL76 are ATM-dependent.

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Figure 3.5. IL-8 induction by UL76 is ATM-dependent. A) 293T cells were transfected with control plasmid or UL76 expression plasmid and cultured in the presence or absence of ATM inhibitor, KU55933 (10 µM). Supernatants were collected 48h post-transfection and IL-8 concentration (pg/ml) was determined by ELISA. Data are expressed as fold induction to control vector (means ± SD of triplicate wells) from one of three similar experiments. Expression of UL76 was confirmed by western blot (below) and β-actin was used as loading control. B) ATM -/- cells were transfected with control plasmid or UL76 expression plasmid. Supernatants were collected 48h post-transfection and IL-8 concentration (pg/ml) was determined by ELISA. Cells were stimulated for 5h with etoposide (10 µM) or TNF α (20 ng/ml). Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Expression of UL76 was confirmed by western blot (below) and β-actin was used as loading control.

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3.4.5. UL76 endonuclease motifs mutation reduced IL-8 induction and has no effect on cell cycle arrest

There is clear evidence that UL76 activates the DNA damage response based on the increased numbers of DNA double stranded breaks and the phosphorylation of γH2AX (Siew et al. , 2009), ATM and p53 at serine 15 in cells expressing UL76 (Nascimento, unpublished results). The mechanism employed by UL76 for this activation, however, is still unknown. The prediction that the UL24 gene family encodes a novel PD-(D/E)XK endonuclease is a possible explanation (Knizewski et al. , 2006). Comparison of UL24 gene family sequences identified the three conserved PD-(D/E)XK signature amino acids of the endonuclease motif (highlighted in black and indicated by red arrows) which are conserved in all homologues (Fig 3.6A) (Knizewski 2006). An UL76 mutant gene which has these three critical amino acids substituted by a glycine amino acid was used to analyze the impact of the putative endonuclease activity on IL-8 induction. Levels of IL- 8 secreted by cells expressing the mutant UL76 gene were reduced when compared to wild type UL76 gene; however, they were still significantly higher than control vector-expressing cells (Fig 3.6B). Previously, it has been demonstrated that UL24 homologues induce cell cycle arrest (Nascimento et al. , 2009). This modulation of cell cycle by UL24 gene family is also the result of the DNA damage initiated checkpoint activation (Nascimento, unpublished work). Thus, the impact of the putative endonulease motifs in the cell cycle arrest was also explored. As shown in Fig 3.6C, expression of the UL76 mutant gene resulted in cell cycle arrest in G2/M phase, similarly to the wild type UL76 gene. In

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Chapter 3 conclusion, the IL-8 induction and cell cycle arrest by UL76 is not due to the putative endonuclease activity.

.

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Figure 3.6. Putative endonuclease activity impact on UL76 functions. A) Multiple sequence alignment for selected UL24 family representatives (adapted from Knizewski 2006). Conserved PD-(D/E)XK signature amino acids, highlighted in black and indicated by red arrows were mutated to glycine. B) Putative endonuclease motifs mutation reduced induction of IL-8. 293T cells were co-transfected with pcDNA3.1, pcDNA3.1HA-UL76 or pcDNA3.1HA-E/K mut plasmids, IL-8 luciferase reporter and β- Galactosidase plasmid. Luciferase activity was measured in cell lysates at 28h-30h post-transfection. Luciferase activity was normalized to β- Galactosidase activity as a control of transfection efficiency. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Statistical significance is represented as *** p<0.001 . C) Putative endonuclease activity has no effect on cell cycle arrest. 293T cells were transduced with control, UL76 or E/K mut recombinant lentiviruses and cell cycle analyses was performed at 48h post- transduction.

3.5. Discussion

Interleukin-8 is a proinflammatory chemokine induced by several different stimuli, including viral infections such as HCMV (Murayama et al. , 1997). The identification of UL76, a conserved non-homologous HCMV gene belonging to the UL24 gene family, as an IL-8-inducing viral gene (chapter 2) promoted a more detailed study of the mechanism underlying this function.

The IL-8 promoter region contains binding sites for the transcription factors NF-kB, AP-1 and NF-IL-6 and IL-8 gene expression is tightly regulated at transcriptional level. In order to evaluate which transcription factors are involved in IL-8 induction by UL76, a series of deletion mutant luciferase constructs were used in luciferase reporter assays. The fact that IL-8 induction was drastically reduced in the absence of a NF-kB binding site, but not when the AP-1 or NF-IL-6 sites were mutated, indicates that UL76 up-

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Chapter 3 regulates IL-8 expression essentially through NF-kB pathway. The slightly decreased induction of IL-8 observed with the AP-1 or NF- IL-6 binding site mutated and the incomplete abrogation of IL-8 activation in the absence of NF-kB, however, suggest that AP-1 or NF-IL-6 may be required for optimal IL-8 induction in cells expressing UL76. These results are consistent with other reports which, collectively, demonstrate that the NF-kB site is required for IL-8 induction by most stimuli and in the majority of cell types studied. Although AP-1 or NF-IL-6 sites usually contribute for maximal IL-8 activation, they are not always necessary, depending on the cell type (Hoffmann et al. , 2002). Further characterization of NF-kB activation by UL76 indicated that it induces IL-8 through the NF-kB canonical pathway. This was demonstrated by the requirement of functional IKK β and the degradation of the IkB protein for the observed IL-8 activation by UL76. Moreover, expression of UL76 promoted the translocation of p65 subunit to the nucleus. These events, however, typically occur in the cytoplasm and are usually triggered by membrane-receptor stimulation. Thus, the exact mechanism of how UL76 activates the NF-kB canonical pathway is an interesting paradox as UL76 is a nuclear protein. In recent years, however, research has identified alternative mechanisms leading to NF-kB pathway activation. One of these pathways is induced by activation of DNA damage and, in contrast to inflammatory stimuli, the signal originates in the nucleus. Since it has been shown that UL76 is able to induce double strand breaks and, consequently, activate DNA damage (Siew et al. , 2009), our hypothesis was that UL76 induced IL-8 expression through activation of DNA damage. Activation of NF-kB by genotoxic stress involves not only activation of ATM but also a series of post-translational modifications of nuclear NEMO protein.

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Consistent with this pathway, expression of UL76 resulted in an accumulation of NEMO in the nucleus and in its phosphorylation at serine 85. A similar NF-kB activation occurs in response to genotoxic stress but not with extracellular stimuli such as TNF α or IL-1β. Finally, the critical role of ATM in IL-8 induction by UL76 was demonstrated in experiments using a specific ATM inhibitor and an ATM knockout cell line. Abrogation of IL-8 induction by UL76 occurred with both approaches.

Interestingly, previous work from our group demonstrated that UL76, as other human and murine UL24 homologues, induces G2/M cell cycle arrest and an associated phosphorylation of p53 and downstream increase in p21 expression (Nascimento et al. , 2009; Nascimento & Parkhouse, 2007). The fact that UL76 is able to activate DNA damage provides a possible basis for the observed cell cycle arrest.

A promising clue was the identification of the UL24 gene family as a novel putative PD-(D/E)XK endonuclease (Knizewski et al. , 2006). The existing classification of PD-(D/E)XK endonuclease structures includes a number of families such as several restriction endonucleases (EcoRI, EcoRII, BamHI, BglI, Cfr10I, NaeI,etc.), DNA repair enzymes (MutH and Vsr), Holliday junction resolvases (Hjc and Hje), and other nucleotide-cleaving enzymes (Murzin et al. , 1995). Thus, the DNA damage activation by UL76 could be a direct effect of its endonuclease activity. Mutation of the three predicted endonuclease motifs resulted in a reduction of IL-8 induction but still significantly higher than control vector. The putative endonuclease motifs are included in the sequence region domains that are conserved in all UL24 homologues suggesting that they have a critical role in this gene family function. The

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Chapter 3 reduction of IL-8 induction may be due to the disruption of these conserved domains and not related to a putative endonuclease activity. Similarly, the UL76 mutant gene was still able to induce the cell cycle arrest already demonstrated for the wild type UL76 gene (Nascimento et al. , 2009). It is possible that the bioinformatically predicted endonuclease motifs are not related to a real endonuclease activity and, indeed, no endonuclease activity has been demonstrated experimentally for any of the UL24 homologues.

In conclusion, UL76 is a multi-functional protein that is able to modulate not only the cell cycle but also induce the expression of IL-8. Both functions are clearly beneficial to HCMV infection.

3.6. Acknowledgments

The endonuclease mutant UL76 gene construction and cell cycle analysis was performed by Rute Nascimento.

3.7. References

Aihara, M., Tsuchimoto, D., Takizawa, H., Azuma, A., Wakebe, H., Ohmoto, Y., Imagawa, K., Kikuchi, M., Mukaida, N. & Matsushima, K. (1997). Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45. Infect Immun 65 , 3218-3224. Baggiolini, M. & Clark-Lewis, I. (1992). Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett 307 , 97-101. Brockman, J. A., Scherer, D. C., McKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y. & Ballard, D. W. (1995). Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. Mol Cell Biol 15 , 2809-2818. Cota, M., Kleinschmidt, A., Ceccherini-Silberstein, F., Aloisi, F., Mengozzi, M., Mantovani, A., Brack-Werner, R. & Poli, G. (2000). Upregulated expression of interleukin-8, RANTES and

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chemokine receptors in human astrocytic cells infected with HIV- 1. J Neurovirol 6, 75-83. Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H. & Kracht, M. (2002). Multiple control of interleukin-8 gene expression. J Leukoc Biol 72 , 847-855. Kasahara, T., Mukaida, N., Yamashita, K., Yagisawa, H., Akahoshi, T. & Matsushima, K. (1991). IL-1 and TNF-alpha induction of IL-8 and monocyte chemotactic and activating factor (MCAF) mRNA expression in a human astrocytoma cell line. Immunology 74 , 60- 67. Knizewski, L., Kinch, L., Grishin, N. V., Rychlewski, L. & Ginalski, K. (2006). Human herpesvirus 1 UL24 gene encodes a potential PD- (D/E)XK endonuclease. J Virol 80 , 2575-2577. Medin, C. L. & Rothman, A. L. (2006). Cell type-specific mechanisms of interleukin-8 induction by dengue virus and differential response to drug treatment. J Infect Dis 193 , 1070-1077. Miyamoto, S. (2011). Nuclear initiated NF-κB signaling: NEMO and ATM take center stage. Cell Res 21 , 116-130. Murayama, T., Ohara, Y., Obuchi, M., Khabar, K. S., Higashi, H., Mukaida, N. & Matsushima, K. (1997). Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB- binding sites of the interleukin-8 gene. J Virol 71 , 5692-5695. Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia, C. (1995). SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 247 , 536-540. Nascimento, R., Dias, J. D. & Parkhouse, R. M. (2009). The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex. Arch Virol 154 , 1143-1149. Nascimento, R. & Parkhouse, R. M. (2007). Murine gammaherpesvirus 68 ORF20 induces cell-cycle arrest in G2 by inhibiting the Cdc2- cyclin B complex. J Gen Virol 88 , 1446-1453. Perkins, N. D. (2007). Integrating cell-signalling pathways with NF- kappaB and IKK function. Nat Rev Mol Cell Biol 8, 49-62. Siew, V. K., Duh, C. Y. & Wang, S. K. (2009). Human cytomegalovirus UL76 induces chromosome aberrations. J Biomed Sci 16 , 107. Wu, Z. H., Shi, Y., Tibbetts, R. S. & Miyamoto, S. (2006). Molecular linkage between the kinase ATM and NF-kappaB signaling in response to genotoxic stimuli. Science 311 , 1141-1146.

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Zandi, E., Chen, Y. & Karin, M. (1998). Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: discrimination between free and NF- kappaB-bound substrate. Science 281 , 1360-1363.

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CHAPTER 4

MECHANISM OF IL-8 INDUCTION BY HUMAN

CYTOMEGALOVIRUS AND IMPACT OF THE UL76 GENE

Chapter 4

4.1. Summary

Human cytomegalovirus has evolved a broad range of host evasion strategies, modulating not only innate and adaptive immunity but also host cell biology, for example, the cell cycle and apoptosis. The observed induction of the proinflammatory chemokine IL-8 during HCMV infection is particularly important for viral replication and possibly contributes to the efficient dissemination of the virus by neutrophils (Craigen et al. , 1997; Murayama et al. , 1997). In Chapter 2, UL76 was identified as a gene inducing IL-8. To evaluate the importance of UL76 gene in the induction of IL-8 during viral infection, the impact of a UL76 deletion mutant on the expression of IL-8 was assessed. In contrast to the rapid induction of IL-8 in human fibroblasts infected with wild type AD169 BAC HCMV, cells infected with the UL76 mutant virus secreted decreased levels of IL-8. This result indicates that UL76 has a significant role in the induction of IL-8 during HCMV infection. Based on the fact that UL76 induces IL-8 by activating NF-kB through DNA damage, the role of ATM in the IL-8 induction during HCMV infection was explored. Significantly, the levels of IL-8 secreted by ATM -/- cells when infected with HCMV were drastically reduced when compared to wild type human fibroblasts. Collectively, these results suggest that IL-8 induction as result of DNA damage and NF-kB activation by UL76 has an important role during HCMV infection.

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4.2. Introduction

Human cytomegalovirus, as a β-herpesvirus, is characterized by its slow replication cycle and limited cell tropism (Mocarski et al. , 2007). During the different stages of the life cycle, HCMV impacts on a wide range of host cell gene expression associated with several cellular responses such as modulation of cell cycle or activation of transcription factors and cytokines (Evers et al. , 2004).

The proinflammatory chemokine IL-8 is one example of the genes up-regulated in different cell types upon infection with HCMV (Craigen et al. , 1997; Grundy et al. , 1998; Murayama et al. , 1997). Moreover, addition of IL-8 to cells infected with HCMV enhances viral replication and virion production (Murayama et al. , 1994). The primary role of IL-8 as chemoattractant for neutrophils has been shown to be relevant during HCMV infection. The proposed model is that HCMV-infected endothelial cells, in addition to other infected cells in the tissues, recruit neutrophils to sites of infection by their secretion of IL-8. The infected endothelial cells then transmit the virus to the neutrophils, which disseminate the virus throughout the body via the bloodstream (Craigen et al. , 1997; Grundy et al. , 1998; Murayama et al. , 1997)). The mechanism of HCMV-induced IL-8 is still not clear, although deletion mutant analysis of IL-8 promoter indicated a critical role for the NF-kB and AP-1 transcription factors (Murayama et al. , 1997).

Significantly, HCMV replication activates ATM, a key kinase of the DNA damage response. The activation of the checkpoint pathway that responds to double strand DNA breaks by HCMV infection, however, is blocked downstream at the level of the effector checkpoint kinase 2 (Chk2) (Gaspar & Shenk, 2006; Luo et al. ,

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2007). Several viruses, such as HIV-1, require activation of ATM for an efficient replication (Ariumi & Trono, 2006). Recently, it has been demonstrated that activation of ATM is also necessary for replication of HCMV, with H2AX, an ATM downstream target, and E2F1 transcription factor contributing to the efficient replication of the virus (E et al. , 2011). Thus, activation of the DNA damage response is essential for the replication of HCMV, but the signaling pathway is blocked, by mislocalization of checkpoint proteins, preventing the complete activation of the checkpoint pathway (Gaspar & Shenk, 2006; Luo et al. , 2007).

In previous chapters, it has been demonstrated that the HCMV UL76 gene induces IL-8 expression through an ATM-dependent activation of NF-kB. In this chapter, the role of UL76 and ATM in IL- 8 induction during HCMV infection is confirmed. Thus, reduced levels of IL-8 were observed after (1) infection of normal human fibroblasts with an UL76 mutant virus or (2) after infecting an ATM deficient cell line with HCMV wild type virus.

4.3. Materials and Methods

4.3.1. Cells

Human foreskin fibroblasts (HFF) (obtained from European Collection of Cell Cultures) and A-T human fibroblast cell line (GM09607 ATM-/-) (obtained from the Coriell Institute for Medical Research) were cultured in Minimum Essential Medium with Earle's salts supplemented with 10% fetal calf serum (Gibco).

4.3.2. HCMV stock production

The HCMV laboratory strain AD169 bacterial artificial chromosome (BAC) DNA was obtained from Dr. Koszinowski. The UL76 mutant 101

Chapter 4 virus (TNUL76), a gift from Dr Shenk, was generated by site- directed transposon mutagenesis of HCMV AD169 BAC and has been previously described (Yu et al. , 2003). Wild type or UL76 mutant virus BAC DNA was transfected in HFF cells by electroporation. Supernatants of transfected cells were collected and used for virus stock production. To prepare virus stocks of wild type AD169 virus and TNUL76 mutant virus, HFF cells were infected at a multiplicity of infection (MOI) of 0.01. After virus adsorption for one hour, infected cells were cultured at 37ºC and medium was collected every three days. Pre-cleared supernatants were centrifuged two hours at 12000 rpm at room temperature. Virus aliquots were stored at -80ºC. Virus stock titers were determined by plaque assay. Briefly, HFF cells were cultured with 10-fold dilutions of virus suspension and allowed to absorb for 1h. Cells were then cultured with complete medium containing 10% carboxymethylcellulose (CMC) for 10-15 days. Cellular monolayers were fixed in 4% paraformaldehyde and stained with 0.1% toluidine blue. Quantification of the viral plaques was performed using a dissecting microscope.

4.3.3. Enzyme-linked Immunoabsorbent Assay (ELISA)

HFF or ATM -/-cells were infected with HCMV at a MOI of 3 or mock infected. After incubation for one hour to allow virus adsorption, cells were incubated in culture medium for the indicated times. Supernatants were harvested and clarified by centrifugation. The concentration of secreted IL-8 was determined by ELISA following the manufacturer’s protocol (IL-8 ELISA kit, BD Biosciences). Plates were analyzed at 450 nm using a BioRad ELISA Reader (BioRad) and levels of IL-8 were determined by comparison to a standard curve.

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4.3.4. Western blot

Infected cells used in ELISA experiments were lysed and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories). After blocking with 5% nonfat milk for one hour at room temperature, membranes were incubated with a monoclonal antibody for anti-CMV IE1 (Santa Cruz Biotechnology, Inc) followed by horseradish peroxidase-conjugated anti-mouse secondary antibody (Sigma). As loading control, we used a horseradish peroxidase-conjugated anti- β-actin antibody (Sigma). Immunoblots were developed by enhanced chemiluminescence detection according to the manufacturer's instructions (ECL, Amersham Pharmacia Biotech).

4.4. Results

4.4.1. Induction of IL-8 by HCMV is reduced in the absence of UL76

In the previous chapters it was demonstrated that HCMV gene UL76 is able to stimulate IL-8 expression at both transcriptional and protein levels. In order to evaluate the impact of UL76 on up- regulation of IL-8 in the context of HCMV infection, we used an UL76 transposon mutant HCMV previously described (Yu et al. , 2003). Supernatants from human fibroblasts infected with wild type HCMV AD169 strain or UL76 mutant virus (TNUL76) were collected at the indicated time points and IL-8 concentration was determined by ELISA. Consistent with previous studies, HCMV infection resulted in high levels of IL-8 secretion during the course of the experiment (Fig. 4.1). Induction of IL-8 in cells infected with the UL76 mutant virus, however, was significantly reduced.

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Infection with each virus was confirmed by the presence of the viral protein IE1 and β-actin was used as loading control. Thus, UL76 is essential for optimal induction of IL-8 by HCMV.

Figure 4.1. Requirement of UL76 for optimal IL-8 induction by HCMV. HFF cells were infected with HCMV AD169 or UL76 mutant HCMV (TNUL76) at a MOI of 3. Supernatants were collected at 24h, 48h and 72h hours post-infection and IL-8 concentration was determined by ELISA. Data are expressed as means ± SD of triplicate wells from one of three similar experiments. Cells were lysed at 72h post-infection and viral infection was confirmed by western blot (below) using an anti-IE1 antibody for the CMV viral protein and tubulin as loading control.

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4.4.2. ATM is required for IL-8 induction by HCMV

Deletion mutant analysis of IL-8 promoter indicated that AP-1 and NF-kB transcription factors were involved in IL-8 induction by HCMV (Murayama et al., 1997). The mechanism used by HCMV to induce IL-8 expression, however, is still not clear. In previous chapters it has been described how HCMV UL76 gene induces IL-8 through the activation of NF-kB pathway in an ATM-dependent manner. As UL76 is required for maximal IL-8 induction by HCMV (Fig.4.1) and HCMV infection activates ATM (Gaspar & Shenk, 2006; Luo et al. , 2007), we hypothesized that ATM would also have a role in IL-8 up-regulation during viral infection. To test this hypothesis an ATM -/- fibroblast cell line was infected with HCMV AD169. Supernatants were collected at the indicated time points and IL-8 concentration was determined by ELISA. As shown in Figure 4.2., IL-8 induction by HCMV is significantly reduced in the absence of ATM. Infection was confirmed by the presence of the viral protein IE1 (Fig. 4.2). Collectively, these results indicate that during HCMV infection, UL76, and possibly other gene(s), induces IL-8 expression, at least in part, through activation of ATM.

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Figure 4.2. IL-8 induction by HCMV is ATM-dependent. ATM -/- cells were infected with HCMV AD169 at a MOI of 3. Supernatants were collected at 24h, 48h and 72h hours post-infection and IL-8 concentration was determined by ELISA. Data are expressed as mean ± SD and are representative of three similar experiments. Cells were lysed at 72h post- infection and viral infection was confirmed by western blot (below) using an anti-IE1 antibody for the CMV viral protein and tubulin as loading control.

4.5. Discussion

Induction of the proinflammatory chemokine IL-8 during HCMV infection is particularly important for viral replication and virion production, and also contributes to the efficient dissemination of the virus by neutrophils (Craigen et al. , 1997; Murayama et al. , 1997). In previous chapters, UL76 has been characterized as a gene

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Chapter 4 inducing IL-8. In order to determine its role in the context of viral infection, we used an UL76 mutant virus (TNUL76), generated by site-directed transposon mutagenesis of the HCMV AD169 strain (Yu et al. , 2003). Comparison of IL-8 secretion in cells infected with wild type HCMV or UL76 mutant virus showed a significant reduction of secreted IL-8 in the absence of UL76. This result indicates that UL76 has a critical role in the up-regulation of IL-8 during HCMV infection. The fact that cells infected with the UL76 mutant virus still produce higher levels of IL-8 compared to mock infected cells suggests, however, that there may be other gene(s) which contribute to induction of IL-8 by HCMV. The only HCMV gene that has been described as IL-8 activator until now, is the immediate early 1 gene (IE1) (Murayama et al. , 1997). Thus, the incomplete inhibition of IL-8 secretion by HCMV observed in the absence of UL76 is possibly due to the effect of IE1 gene. Conversely, the partial inhibition of HCMV-induced IL-8 expression at transcriptional and protein levels when IE1 expression was blocked (Cinatl et al. , 2000) may be explained by the presence of UL76. The existence of other gene(s) that may also contribute for HCMV-induced IL-8 expression, in addition to IE1 and UL76, is not excluded. Interestingly, HCMV UL146 gene encodes vCXCL1, a homologue to CXC chemokine genes, which functions as a selective agonist for CXCR2 and, with lower affinity, for CXCR1 (Lüttichau, 2010; Penfold et al. , 1999). In this work, however, the HCMV AD169 strain which has several genes deleted, including UL146 was used. Thus, the IL-8 production observed in cells infected with wild type HCMV or UL76 mutant virus is not dependent on the presence of the viral CXCL1. Whether the mechanism of induction of IL-8 by the intact virus is exactly the same as that described for the UL76 gene alone

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(chapter 3) was not clear. Although both UL76 alone and HCMV activate the NF-kB pathway to induce IL-8 expression, the AP-1 transcription factor is also required in HCMV infected cells (Murayama et al. , 1997). The work described in chapter 3 suggested that ATM would also have a role in IL-8 up-regulation during viral infection, and indeed infection of ATM -/- cells with wild type HCMV resulted in lower levels of secreted IL-8 compared to the similar infection of normal human fibroblasts. The fact that the inhibition was not complete with the UL76 deletion mutant virus, nor with the ATM negative cells is relevant. Essentially, we may conclude that HCMV gene(s) other than UL76 have evolved to ensure the induction of IL-8, but it is probable, that the mechanism of such additional genes is not identical to that employed by UL76.

Interestingly, both UL76 and ATM have a positive effect on HCMV replication. The HCMV AD169 strain functional map analysis that described the TNUL76 mutant virus used in this chapter, identified UL76 as one of the 27 augmenting genes. Infection of human fibroblasts with TNUL76 virus resulted in a small plaque phenotype and a significant reduction of viral replication compared to wild type virus (Yu et al. , 2003). On the other hand, the importance of ATM for HCMV replication was recently described. The authors demonstrated that HCMV replication was compromised when ATM was inhibited or depleted using a specific inhibitor, siRNA or a knock out cell line (E et al. , 2011). Based on the decreased expression of IL-8 demonstrated above in the absence of UL76 or ATM, it is possible that the observed defect in viral replication is associated with the reduced IL-8 levels. Supporting this hypothesis is the fact that IL-8 enhances HCMV replication (Murayama et al. ,

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1994). Thus, it would be interesting to explore the effect of exogenous IL-8 on replication of the UL76 mutant virus.

4.6. References

Ariumi, Y. & Trono, D. (2006). Ataxia-telangiectasia-mutated (ATM) protein can enhance human immunodeficiency virus type 1 replication by stimulating Rev function. J Virol 80 , 2445-2452. Cinatl, J., Kotchetkov, R., Weimer, E., Blaheta, R. A., Scholz, M., Vogel, J. U., Gümbel, H. O. & Doerr, H. W. (2000). The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts. J Med Virol 60 , 313-323. Craigen, J. L., Yong, K. L., Jordan, N. J., MacCormac, L. P., Westwick, J., Akbar, A. N. & Grundy, J. E. (1997). Human cytomegalovirus infection up-regulates interleukin-8 gene expression and stimulates neutrophil transendothelial migration. Immunology 92 , 138-145. E, X., Pickering, M. T., Debatis, M., Castillo, J., Lagadinos, A., Wang, S., Lu, S. & Kowalik, T. F. (2011). An E2F1-mediated DNA damage response contributes to the replication of human cytomegalovirus. PLoS Pathog 7, e1001342. Evers, D. L., Wang, X. & Huang, E. S. (2004). Cellular stress and signal transduction responses to human cytomegalovirus infection. Microbes Infect 6, 1084-1093. Gaspar, M. & Shenk, T. (2006). Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins. Proc Natl Acad Sci U S A 103 , 2821-2826. Grundy, J. E., Lawson, K. M., MacCormac, L. P., Fletcher, J. M. & Yong, K. L. (1998). Cytomegalovirus-infected endothelial cells recruit neutrophils by the secretion of C-X-C chemokines and transmit virus by direct neutrophil-endothelial cell contact and during neutrophil transendothelial migration. J Infect Dis 177 , 1465- 1474. Luo, M. H., Rosenke, K., Czornak, K. & Fortunato, E. A. (2007). Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection. J Virol 81 , 1934-1950. Lüttichau, H. R. (2010). The cytomegalovirus UL146 gene product vCXCL1 targets both CXCR1 and CXCR2 as an agonist. J Biol Chem 285 , 9137-9146.

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Mocarski, E. S., Shenk, T. & Pass, R. F. (2007). Cytomegaloviruses. In Fields Virology, 5th Edition . Edited by D. M. Knipe & P. M. Howley: Lippincott Williams & Wilkins. Murayama, T., Kuno, K., Jisaki, F., Obuchi, M., Sakamuro, D., Furukawa, T., Mukaida, N. & Matsushima, K. (1994). Enhancement human cytomegalovirus replication in a human lung fibroblast cell line by interleukin-8. J Virol 68 , 7582-7585. Murayama, T., Ohara, Y., Obuchi, M., Khabar, K. S., Higashi, H., Mukaida, N. & Matsushima, K. (1997). Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB- binding sites of the interleukin-8 gene. J Virol 71 , 5692-5695. Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W. & Schall, T. J. (1999). Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci U S A 96 , 9839- 9844. Yu, D., Silva, M. C. & Shenk, T. (2003). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100 , 12396-12401.

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CHAPTER 5

FINAL CONSIDERATIONS

Chapter 5

5.1. Final Considerations

Herpesviruses are an outstanding example of how pathogens adapt and co-evolve with their hosts. Their genome contains an impressive number of genes manipulating host cell biology and immune responses in order to favour their survival. The typical herpesvirus life cycle, consisting of replicative (lytic) and latent phases, is a challenge for the development of a global antiviral therapy or protective vaccines. A promising approach is to explore new viral targets, particularly viral proteins involved in host evasion. An effective herpesvirus vaccine could therefore be a genetically targeted mutant with one or more non-immunogenic host evasion genes deleted, and with appropriate investigation of the pathogenesis to ensure safety. Several viral proteins involved in immune evasion have already been identified through their homology with cellular proteins. There are, however, evasion proteins encoded by genes without sequence homology to cellular genes that might represent a paradigm of co-evolution, or could simply be examples for which the host homologues have not yet been identified (Alcami & Koszinowski, 2000). In these cases, the viral protein function can only be accessed by functional assays or structural assays, such as x-ray crystallography (Cooray et al. , 2007).

This work focused in one example of these non-homologous genes, the UL24 gene family. Its relevance is emphasized by the fact that is the only core gene, conserved in all herpesviruses, without an assigned function. The lack of homology with any cellular protein, and its identification as a conserved non-essential gene, suggests that it may be involved in host evasion mechanisms, and indeed it has been recently shown that UL24

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Chapter 5 homologues from the three herpesviruses subfamilies induce cell cycle arrest at G2/M phase (Nascimento et al. , 2009).

Induction of IL-8 by UL76 gene

In order to further characterize the intracellular pathways that are modulated by UL24 gene family, microarray analysis was performed in cells expressing the murine homologue, ORF20 from MHV-68, and the human homologue from HCMV, UL76 gene. One of the genes that was up-regulated by UL76, but not by ORF20, was interleukin-8, a proinflammatory chemokine primarily a chemoattractant for neutrophils, but also involved in angiogenesis and inhibition of interferon responses. The fact that ORF20 had no impact on IL-8 expression may be related to the specific life cycle of the virus or the herpesvirus subfamily, since MHV-68 is a γ- herpesvirus and HCMV is a β-herpesvirus. On the other hand, the inability of ORF20 to induce IL-8 may be related to the fact that there is no known murine IL-8 homologue. Thus, the murine UL24 homologue possibly modulates the IL-8 functional homologues, such as KC or MIP-2, which are also CXC chemokines and attract neutrophils (Bozic et al. , 1995; Driscoll et al. , 1995). Studies with the murine herpesvirus support this hypothesis as increased levels of KC and MIP-2 were detected in mice infected with MHV-68 (Sarawar et al. , 2002), and KC was shown to enhance MHV-68 replication in permissive fibroblasts (Lee et al. , 2003). As this function has been described for UL76 from HCMV, MCMV infection of mice would be other important model to study the role of UL76 (m76 in MCMV) in the regulation of these chemokines in vivo .

This work focused in the mechanism of induction of IL-8 by UL76. Activation of IL-8 by transfection of cells with UL76 was confirmed

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Chapter 5 at both transcriptional and protein levels. Deletion mutant analysis of the IL-8 promoter indicated that activation of the NF-kB pathway was essential for IL-8 induction by UL76. Analysis of cellular proteins involved in NF-kB activation such as IKK β, IkB α and p65 demonstrated that UL76 induces IL-8 through the NF-kB canonical pathway. Other viral proteins such as KSHV-encoded viral FLICE inhibitory protein K13 (Sun et al. , 2006), Vpr from HIV-1 (Roux et al. , 2000) or latent membrane protein-1 (LMP1) from EBV (Yoshizaki et al. , 2001) have been identified as being able to induce IL-8 expression as a result of NF-kB activation. However, little is known about the upstream events that lead to NF-kB pathway activation and culminate with IL-8 induction. In this work, activation of NF-kB and subsequent induction of IL-8 result from the activation of the DNA damage response. Generally, the NF-kB canonical pathway is activated by external stimuli such as proinflammatory cytokines. Recently, however, an atypical pathway has been characterized as a result of genotoxic stress. The involvement of this pathway in IL-8 induction by UL76 was suggested by the fact that UL76 activates the DNA damage response, and is consistent with its nuclear localization. Similarly, the cell cycle arrest observed in cells expressing UL76 results from the activation of DNA damage checkpoint at the G2 phase (Nascimento, unpublished work). Thus, this work identified a novel function of UL76 in addition to cell cycle modulation, with both effects resulting from the activation of the DNA damage response. A clue as to exactly how the UL76 gene both manipulate the cell cycle and induce IL-8 was provided by the identification of UL24 gene family as a putative endonuclease (Knizewski et al. , 2006). Disappointingly, however, it was demonstrated that the putative endonuclease motifs, although necessary for optimal IL-8

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Chapter 5 induction, were not essential, neither for IL-8 induction, nor for cell cycle arrest (Chapter 3). In this context, it would be useful to determine experimentally if UL24 homologues do have endonuclease activity. Relevant to this is the mechanism of genome processing and linearization as a necessary step in the virus life cycle, as this requires DNAse activity, whether of cellular or viral origin. It is also not known if the effect of UL76 results from a direct interaction with a protein involved in the DNA damage response as the interacting partner(s) of UL76 has not yet been identified. Given the conservation of the UL24 family in herpesviruses, an immediate question is whether the mechanism we have described for UL76 of HCMV is conserved in the other major human herpesviruses homologues. My preliminary results in cells expressing UL24 from HSV-1 demonstrate that UL24 also induces IL-8 at transcriptional and protein levels suggesting that it has a role in the induction of IL-8 during HSV-1 infection. In fact, induction of IL-8 during HSV-1 infection has been shown in cultures of epithelial cells and keratocytes, and may be associated with the infiltration of neutrophils observed in stromal keratitis induced by HSV-1 (Li et al. , 2006; Oakes et al. , 1993). The impact of other UL24 homologues was not analyzed, but it is clear that IL-8 has a critical role in most herpesviruses such as HHV-6 (Caruso et al. , 2002), varicella-zoster virus (VZV) (Desloges et al. , 2008), KSHV (Lane et al. , 2002) and EBV (Hsu et al. , 2008). Finally, Vpr from HIV-1 is able to induce IL-8 through NF-kB and also promotes cell cycle arrest and the DNA damage response (Nakai-Murakami et al. , 2007; Roux et al. , 2000; Zimmerman et al. , 2004). Thus, it is possible that, similar to UL76, IL-8 induction and

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Chapter 5 cell cycle arrest by vpr are associated with activation of DNA damage.

Induction of IL-8 by HCMV infection

Induction of IL-8 during HCMV infection is particularly important for viral replication and virion production (Murayama et al. , 1994). The accepted hypothesis is that infected endothelial cells produce higher levels of IL-8 that will attract neutrophils to the site of infection. It has been demonstrated that neutrophils can acquire infectious HCMV virions from infected endothelial cells and are able to transmit them to other cell types of different tissues, thus, contributing for HCMV dissemination throughout the body (Penfold et al. , 1999). The importance of IL-8 during HCMV infection is reforced by the fact that the UL146 gene encodes vCXCL1, a homologue of the CXC chemokine genes such as IL-8 (Lüttichau, 2010; Penfold et al. , 1999). The UL146 gene is, however, deleted from several HCMV strains, including the AD169 used in this study and shown to induce IL-8. Thus there must be genes other than UL146 involved in the IL-8 up-regulation. The only HCMV gene that has been described as IL-8 activator, until now, is the Immediate Early 1 gene (IE1) (Murayama et al. , 1997). The fact that infection with an UL76 mutant virus results in decreased levels of IL-8 produced indicates that UL76 has also a critical role in the up- regulation of IL-8 during HCMV infection. Similar to UL76, HCMV activates the NF-kB pathway to induce IL-8 expression (Murayama et al. , 1997). In this work it is demonstrated that ATM has a critical role in the induction of IL-8, both in UL76 transfected cells and during HCMV infection.

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The mechanism of IL-8 induction described in this work indicates that the less efficient replication of HCMV in the absence of ATM may be due to the reduced levels of IL-8 observed during viral infection in ATM knockout cells. Thus, the impact of exogenous IL- 8 in the replication of UL76 mutant virus and the wild type virus in ATM knockout cells should be explored in the future. It is interesting to note that either the absence of UL76 or of ATM results in inefficient viral replication (E et al. , 2011; Yu et al. , 2003). The identification of UL76 as an augmenting gene of viral growth indicates that its function, although not essential, is important for efficient HCMV replication. This may be related to the up-regulation of IL-8 described in this work as it has been shown that IL-8 enhances HCMV replication and virion production. Similarly, several recent studies demonstrated that activation of the DNA damage response is essential for replication of different viruses such as polyomavirus (Dahl et al. , 2005), parvovirus (Luo et al. , 2011), human papillomavirus (HPV) (Moody & Laimins, 2009) and HIV-1 (Ariumi & Trono, 2006). Thus different viruses have evolved different modes for manipulating the DNA Damage response. It is still not entirely clear how virus-induced DNA damage is beneficial to viral replication, or whether it may be viewed as a “trade off” due to the requirement for DNAse activity in the virus life cycle. Similarly unclear, is whether the virus mediated initiation of the DNA Damage response involves the recognition of existing double strand breaks or results from the recruitment of repair factors to viral replication centers.

In summary, the non-homologous UL76 gene of HCMV has evolved for manipulation of the host cell cycle, and also activates

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5.2. References Alcami, A. & Koszinowski, U. H. (2000). Viral mechanisms of immune evasion. Trends Microbiol 8, 410-418. Ariumi, Y. & Trono, D. (2006). Ataxia-telangiectasia-mutated (ATM) protein can enhance human immunodeficiency virus type 1 replication by stimulating Rev function. J Virol 80 , 2445-2452. Bozic, C. R., Kolakowski, L. F., Gerard, N. P., Garcia-Rodriguez, C., von Uexkull-Guldenband, C., Conklyn, M. J., Breslow, R., Showell, H. J. & Gerard, C. (1995). Expression and biologic characterization of the murine chemokine KC. J Immunol 154 , 6048-6057. Caruso, A., Rotola, A., Comar, M., Favilli, F., Galvan, M., Tosetti, M., Campello, C., Caselli, E., Alessandri, G., Grassi, M., Garrafa, E., Cassai, E. & Di Luca, D. (2002). HHV-6 infects human aortic and heart microvascular endothelial cells, increasing their ability to secrete proinflammatory chemokines. J Med Virol 67 , 528-533. Cooray, S., Bahar, M. W., Abrescia, N. G., McVey, C. E., Bartlett, N. W., Chen, R. A., Stuart, D. I., Grimes, J. M. & Smith, G. L. (2007). Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein. J Gen Virol 88 , 1656-1666. Dahl, J., You, J. & Benjamin, T. L. (2005). Induction and utilization of an ATM signaling pathway by polyomavirus. J Virol 79 , 13007-13017. Desloges, N., Schubert, C., Wolff, M. H. & Rahaus, M. (2008). Varicella- zoster virus infection induces the secretion of interleukin-8. Med Microbiol Immunol 197 , 277-284. Driscoll, K. E., Hassenbein, D. G., Howard, B. W., Isfort, R. J., Cody, D., Tindal, M. H., Suchanek, M. & Carter, J. M. (1995). Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol 58 , 359-364. E, X., Pickering, M. T., Debatis, M., Castillo, J., Lagadinos, A., Wang, S., Lu, S. & Kowalik, T. F. (2011). An E2F1-mediated DNA damage response contributes to the replication of human cytomegalovirus. PLoS Pathog 7, e1001342. Hsu, M., Wu, S. Y., Chang, S. S., Su, I. J., Tsai, C. H., Lai, S. J., Shiau, A. L., Takada, K. & Chang, Y. (2008). Epstein-Barr virus lytic transactivator Zta enhances chemotactic activity through

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induction of interleukin-8 in nasopharyngeal carcinoma cells. J Virol 82 , 3679-3688. Knizewski, L., Kinch, L., Grishin, N. V., Rychlewski, L. & Ginalski, K. (2006). Human herpesvirus 1 UL24 gene encodes a potential PD- (D/E)XK endonuclease. J Virol 80 , 2575-2577. Lane, B. R., Liu, J., Bock, P. J., Schols, D., Coffey, M. J., Strieter, R. M., Polverini, P. J. & Markovitz, D. M. (2002). Interleukin-8 and growth-regulated oncogene alpha mediate angiogenesis in Kaposi's sarcoma. J Virol 76 , 11570-11583. Lee, B. J., Koszinowski, U. H., Sarawar, S. R. & Adler, H. (2003). A gammaherpesvirus G protein-coupled receptor homologue is required for increased viral replication in response to chemokines and efficient reactivation from latency. J Immunol 170 , 243-251. Li, H., Zhang, J., Kumar, A., Zheng, M., Atherton, S. S. & Yu, F. S. (2006). Herpes simplex virus 1 infection induces the expression of proinflammatory cytokines, interferons and TLR7 in human corneal epithelial cells. Immunology 117 , 167-176. Luo, Y., Chen, A. Y. & Qiu, J. (2011). Bocavirus infection induces a DNA damage response that facilitates viral DNA replication and mediates cell death. J Virol 85 , 133-145. Lüttichau, H. R. (2010). The cytomegalovirus UL146 gene product vCXCL1 targets both CXCR1 and CXCR2 as an agonist. J Biol Chem 285 , 9137-9146. Moody, C. A. & Laimins, L. A. (2009). Human papillomaviruses activate the ATM DNA damage pathway for viral genome amplification upon differentiation. PLoS Pathog 5, e1000605. Murayama, T., Kuno, K., Jisaki, F., Obuchi, M., Sakamuro, D., Furukawa, T., Mukaida, N. & Matsushima, K. (1994). Enhancement human cytomegalovirus replication in a human lung fibroblast cell line by interleukin-8. J Virol 68 , 7582-7585. Murayama, T., Ohara, Y., Obuchi, M., Khabar, K. S., Higashi, H., Mukaida, N. & Matsushima, K. (1997). Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB- binding sites of the interleukin-8 gene. J Virol 71 , 5692-5695. Nakai-Murakami, C., Shimura, M., Kinomoto, M., Takizawa, Y., Tokunaga, K., Taguchi, T., Hoshino, S., Miyagawa, K., Sata, T., Kurumizaka, H., Yuo, A. & Ishizaka, Y. (2007). HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination. Oncogene 26 , 477-486.

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Nascimento, R., Dias, J. D. & Parkhouse, R. M. (2009). The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex. Arch Virol 154 , 1143-1149. Oakes, J. E., Monteiro, C. A., Cubitt, C. L. & Lausch, R. N. (1993). Induction of interleukin-8 gene expression is associated with herpes simplex virus infection of human corneal keratocytes but not human corneal epithelial cells. J Virol 67 , 4777-4784. Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W. & Schall, T. J. (1999). Cytomegalovirus encodes a potent alpha chemokine. Proc Natl Acad Sci U S A 96 , 9839- 9844. Roux, P., Alfieri, C., Hrimech, M., Cohen, E. A. & Tanner, J. E. (2000). Activation of transcription factors NF-kappaB and NF-IL-6 by human immunodeficiency virus type 1 protein R (Vpr) induces interleukin-8 expression. J Virol 74 , 4658-4665. Sarawar, S. R., Lee, B. J., Anderson, M., Teng, Y. C., Zuberi, R. & Von Gesjen, S. (2002). Chemokine induction and leukocyte trafficking to the lungs during murine gammaherpesvirus 68 (MHV-68) infection. Virology 293 , 54-62. Sun, Q., Matta, H., Lu, G. & Chaudhary, P. M. (2006). Induction of IL-8 expression by human herpesvirus 8 encoded vFLIP K13 via NF- kappaB activation. Oncogene 25 , 2717-2726. Yoshizaki, T., Horikawa, T., Qing-Chun, R., Wakisaka, N., Takeshita, H., Sheen, T. S., Lee, S. Y., Sato, H. & Furukawa, M. (2001). Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma. Clin Cancer Res 7, 1946-1951. Yu, D., Silva, M. C. & Shenk, T. (2003). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100 , 12396-12401. Zimmerman, E. S., Chen, J., Andersen, J. L., Ardon, O., Dehart, J. L., Blackett, J., Choudhary, S. K., Camerini, D., Nghiem, P. & Planelles, V. (2004). Human immunodeficiency virus type 1 Vpr- mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation. Mol Cell Biol 24 , 9286-9294.

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ANNEX

MICROARRAY ANALYSIS

gene Accession Empty Empty UL76 UL76 fold lower Call CMV CMV change bound Call of FC SNHG3: small nucleolar RNA host gene (non - AJ006835 50,9 P 346,2 P 6,8 5,4 protein coding) 3 KLHL24: kelch -like 24 (Drosophila) AW006750 28,0 P 180,0 P 6,42 5,67 TAF13: TAF13 RNA polymerase II, TATA box NM_005645 24,5 A 147,9 P 6,05 4,46 binding protein (TBP)-associated factor, 18kDa GEM: GTP binding protein overexpressed in NM_005261 61,2 P 330,0 P 5,39 4,92 skeletal muscle KLHL24: kelch -like 24 (Drosophila) AW006750 65,1 P 337,8 P 5,19 4,05 SLC7A11: solute carrier family 7, (c ationic amino NM_014331 86,4 P 447,9 P 5,18 4,68 acid transporter, y+ system) member 11 IL8: interleukin 8 NM_000584 20,5 A 101,4 P 4,94 3,42 Hs.27008.1 AI742901 121,0 P 582,6 P 4,81 4,58 RPL7 /// /// LOC732071: ribosomal protein L7 AL049545 6,8 A 32,0 P 4,73 2,32 C10orf118: chromosome 10 open reading frame NM_018017 45,8 A 207,8 P 4,53 3,81 118 STK17B: serine/threonine kinase 17b (apoptosis - NM_004226 53,8 P 237,7 P 4,42 3,74 inducing) SGK: serum/glucocorticoid regulated kinase NM_005627 127,6 P 552,4 P 4,33 4,04 CTGF: connective tissue growth factor M92934 38,5 P 158,7 P 4,12 3,58 TAC1: tachykinin, precursor 1 (substance K, NM_003182 21,8 A 89,2 P 4,08 3,41 substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma) SKIL: SKI -like oncogene NM_005414 25,7 A 104,2 P 4,06 3,28 FLJ22639 /// LOC728686: hypothetical protein NM_024796 38,4 P 154,7 P 4,03 3,61 FLJ22639 /// hypothetical protein LOC728686 C21orf91: chromosome 21 open reading frame 91 NM_017447 137,5 P 547,5 P 3,98 3,71

ATF3: activating transcription factor 3 NM_001674 306,8 P 1219,2 P 3,97 3,64 PDE4DIP: phosphodiesterase 4D interacting AB042557 19,3 A 75,9 P 3,94 3,14 protein (myomegalin) GABARAPL1: GABA(A) receptor -associated BF125756 102,2 P 387,0 P 3,79 3,24 protein like 1 PRKAB2: protein kinase, AMP -activated, beta 2 NM_005399 86,5 P 327,7 P 3,79 3,53 non-catalytic subunit MGAT4A: mannosyl (alpha -1,3 -)-glycoprotein NM_012214 45,5 P 171,7 P 3,77 3,3 beta-1,4-N-acetylglucosaminyltransferase, isozyme A IDS: iduronate 2 -sulfatase (Hunter syndrome) NM_000202 153,2 P 576,0 P 3,76 3,5 KLHL11: kelch -like 11 (Drosophila) NM_018143 84,1 A 315,0 P 3,75 3,17 RORA: RAR -related orphan receptor A U04897 40,6 P 151,9 P 3,74 2,93 BCL6: B -cell CLL/l ymphoma 6 (zinc finger protein NM_001706 62,7 P 229,9 P 3,67 3,27 51) /// B-cell CLL/lymphoma 6 (zinc finger protein 51) GLS: glutaminase AF097493 96,2 P 351,7 P 3,66 3,29 GPNMB: glycoprotein (transmembrane) nmb NM_002510 28,0 A 102,6 P 3,66 2,75 MAFF: v-maf musculoaponeurotic fibrosarcoma AL021977 23,6 P 86,3 P 3,66 3,22 oncogene homolog F (avian) API5: apoptosis inhibitor 5 AF229253 140,2 P 509,4 P 3,63 3,24 GABARAPL1 /// GABARAPL3: GABA(A) receptor - AF180519 266,2 P 964,5 P 3,62 3,42 associated protein like 1 /// GABA(A) receptors associated protein like 3 HEXIM1: hexamethylene bis -acetamide inducible NM_006460 379,6 P 1312,0 P 3,46 3,24 1 ETV5: ets variant gene 5 (ets -related molecule) NM_004454 31,0 A 106,9 P 3,45 2,94 MAB21L1: mab -21 -like 1 (C. elegans) NM_005584 51,9 P 177,9 P 3,43 3,07 HIST1H4E: histone cluster 1, H4e NM_003545 58,5 A 199,8 P 3,42 2,52 FAM121A: family with sequence similarity 121A W87634 81,6 P 278,0 P 3,41 2,84

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TNFAIP3: tumor necrosis factor, alpha -induced AI738896 54,4 A 184,3 P 3,39 2,84 protein 3 FEM1B: fem -1 homolog b (C. elegans) NM_015322 187,3 P 628,7 P 3,36 3,06 AQP3: aquaporin 3 (Gill blood group) N74607 100,2 P 334,6 P 3,34 3,15 TEX14: testis expressed sequence 14 /// testis NM_031272 48,4 P 161,5 P 3,34 2,93 expressed sequence 14 ARHGEF12: Rho guanine nucleotide exchange AI807672 83,8 A 278,1 P 3,32 2,79 factor (GEF) 12 RORA: RAR -related orphan receptor A L14611 51,7 P 167,5 P 3,24 2,79 ZFX /// ZFY: zinc finger protein, X -linked /// zinc NM_003411 94,0 P 304,8 P 3,24 3,01 finger protein, Y-linked IDS: iduronate 2 -sulfatase (Hunter syndrome) /// BC006170 9,4 A 30,2 P 3,22 2,29 iduronate 2-sulfatase (Hunter syndrome) Full length insert cDNA YH77E09 AA928506 104,1 P 334,0 P 3,21 2,92 KLF4: Kruppel -like factor 4 (gut) BF514079 95,7 P 305,2 P 3,19 2,74 NLRP1: NLR family, pyrin domain containing 1 NM_021730 204,9 P 652,9 P 3,19 2,95 CFH /// CFHR1: complement factor H /// X56210 55,5 P 176,3 P 3,18 2,85 complement factor H-related 1 RIT1: Ras -like without CAAX 1 AF084462 259,6 P 817,1 P 3,15 2,94 DKFZp547G183: hypothetical protein NM_018705 27,1 P 84,9 P 3,13 2,27 DKFZp547G183 HUS1: HUS1 checkpoint homolog (S. pombe) NM_004507 214,0 P 667,7 P 3,12 2,92 EDNRB: endothelin receptor type B NM_003991 33,9 A 105,4 P 3,11 2,69 VCPIP1: valosin containing protein (p97)/p47 NM_025054 51,7 A 159,9 P 3,1 2,64 complex interacting protein 1 MSX2: msh homeobox 2 D31771 257,2 P 794,9 P 3,09 2,78 TROVE2: TROVE domain family, member 2 AK024044 112,8 P 347,5 P 3,08 2,85 GADD4 5B: growth arrest and DNA -damage - AF078077 101,0 P 309,7 P 3,07 2,73 inducible, beta

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HIST1H2AC: histone cluster 1, H2ac AL353759 101,5 P 310,9 P 3,06 2,68 ITCH: itchy homolog E3 ubiquitin protein ligase AB056663 97,9 P 298,9 P 3,05 2,74 (mouse) DNAJC3: D naJ (Hsp40) homolog, subfamily C, NM_006260 97,1 P 294,9 P 3,04 2,78 member 3 MAFG /// LOC644132: v -maf musculoaponeurotic NM_002359 141,3 P 429,1 P 3,04 2,73 fibrosarcoma oncogene homolog G (avian) /// similar to v-maf musculoaponeurotic fibrosarcoma oncogene family, protein G ELK4: ELK4, ETS -domain protein (SRF accessory NM_001973 52,3 A 158,3 P 3,03 2,63 protein 1) GADD45B: growth arrest and DNA -damage - AF087853 162,8 P 493,2 P 3,03 2,6 inducible, beta Transcribed locus AV764378 119,4 P 362,0 P 3,03 2,72 HBEGF: heparin -binding EGF -like growth factor NM_001945 96,4 P 290,6 P 3,02 2,78 ARL4D: ADP -ribosylation factor -like 4D U25771 133,5 P 393,3 P 2,95 2,67 SNX16: sorting nexin 16 NM_022133 48,8 P 143,4 P 2,94 2,53 CBLL1: Cas -Br -M (murine) ecotropic r etroviral NM_024814 66,9 P 195,6 P 2,93 2,47 transforming sequence-like 1 ABHD5: abhydrolase domain containing 5 NM_016006 153,0 P 446,3 P 2,92 2,73 FGFR1OP /// C9orf4: FGFR1 oncogene partner /// NM_007045 300,6 P 877,9 P 2,92 2,77 chromosome 9 open reading frame 4 SGCB: sarcoglycan, beta (43kDa dystrophin - U29586 435,6 P 1267,0 P 2,91 2,75 associated glycoprotein) SGPL1: sphingosine -1-phosphate lyase 1 NM_003901 63,6 P 182,9 P 2,87 2,55 DNAJB2: DnaJ (Hsp40) homolog, subfamily B, NM_006736 71,8 A 204,8 P 2,85 2,58 member 2 HIST1H2BG /// HIST1H2BC: histone cluster 1, NM_003526 17,0 A 48,2 P 2,84 2,15 H2bg /// histone cluster 1, H2bc TRIM52: tripartite motif -containing 52 AA205660 103,4 P 293,4 P 2,84 2,49

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IDS: iduronate 2 -sulfatase (Hunter syndrome) NM_006123 188,2 P 532,6 P 2,83 2,6 MTRF1L: Mitochondrial translational release AL049992 16,6 A 46,8 P 2,83 2,23 factor 1-like PPP1R15A: protein phosphatase 1, regulatory NM_014330 83,0 P 234,5 P 2,83 2,54 (inhibitor) subunit 15A Transcribed locus, moderat ely similar to AW665096 73,4 A 207,3 P 2,83 2,45 NP_001020307.1 lineage protein 1 [Rattus norvegicus] RSL1D1: ribosomal L1 domain containing 1 AK025446 47,2 M 132,9 P 2,82 2,43 ITCH: itchy homolog E3 ubiquitin protein ligase AL109923 156,0 P 437,8 P 2,81 2,52 (mouse) SMAD7: SMAD family member 7 NM_005904 108,9 P 302,8 P 2,78 2,43 TFPI: tissue factor pathway inhibitor (lipoprotein - AF021834 61,6 P 171,0 P 2,77 2,42 associated coagulation inhibitor) KLF5: Kruppel -like factor 5 (intestinal) AF132818 105,7 P 292,2 P 2,76 2,49 KPNA4: karyopherin alpha 4 (importin alpha 3) U93240 244,1 P 674,4 P 2,76 2,53 CD55: CD55 molecule, decay accelerating factor BC001288 352,3 P 965,5 P 2,74 2,51 for complement (Cromer blood group) CDKN1A: cyclin -dependent kinase inh ibitor 1A NM_000389 258,0 P 703,4 P 2,73 2,37 (p21, Cip1) ABCF2: ATP -binding cassette, sub -family F AL037534 137,4 P 373,3 P 2,72 2,49 (GCN20), member 2 HNRPD: Heterogeneous nuclear W74620 98,5 P 268,4 P 2,72 2,43 ribonucleoprotein D (AU-rich element RNA binding protein 1, 37kDa) PERP: PERP, TP53 apoptosis effector AJ251830 433,4 P 1177,1 P 2,72 2,31 GADD45B: growth arrest and DNA -damage - NM_015675 163,7 P 443,0 P 2,71 2,36 inducible, beta GTF3C4: general transcription factor IIIC, NM_012204 343,3 P 931,7 P 2,71 2,6 polypeptide 4, 90kDa NR4A2: nuclear receptor subfamily 4, group A, S77154 24,4 A 66,2 P 2,71 2,04

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member 2 RNMT: RNA (guanine -7-) methyltransferase AB020966 50,7 P 137,3 P 2,71 2,54 VAMP1: vesicle -associated membrane protein 1 AU150319 98,8 P 267,9 P 2,71 2,36 (synaptobrevin 1) ELL2: elongation factor, RNA polymerase II, 2 NM_012081 147,0 P 396,1 P 2,7 2,48 HBP1: HMG -box transcription factor 1 AF019214 156,2 P 421,1 P 2,7 2,39 AMBN: ameloblastin (enamel matrix protein) NM_016519 83,3 P 224,4 P 2,69 2,44 DGKE: diacylglycerol kinase, epsilon 64kDa NM_003647 34,8 P 93,8 P 2,69 2,23 EIF5A2: eukaryotic translation initiation factor NM_020390 71,7 P 191,9 P 2,68 2,4 5A2 LUM: lumican NM_002345 10,2 A 27,3 P 2,67 2,07 TNFRSF10D: tumor necrosi s factor receptor AF021233 39,6 A 105,8 P 2,67 2,21 superfamily, member 10d, decoy with truncated death domain FUBP1: far upstream element (FUSE) binding AA156865 149,7 P 398,1 P 2,66 2,47 protein 1 KLF4: Kruppel -like factor 4 (gut) AF105036 44,7 A 117,7 P 2,63 2,3 MGC14376: hypothetical protein MGC14376 AF070569 74,0 A 193,7 P 2,62 2,21 CPD: carboxypeptidase D D85390 221,6 P 578,3 P 2,61 2,39 FOS: v -fos FBJ murine osteosarcoma viral BC004490 32,1 A 83,6 P 2,61 2,2 oncogene homolog ALDH1A3: Aldehyde d ehydrogenase 1 family, BG475299 62,4 P 161,7 P 2,59 2,28 member A3 GABARAPL1: GABA(A) receptor -associated BF125756 117,3 P 304,1 P 2,59 2,31 protein like 1 RNASE4: ribonuclease, RNase A family, 4 NM_002937 54,8 P 141,1 P 2,58 2,29 BCL2L11: BCL2 -like 11 (apoptosis facilitator) NM_006538 26,2 A 67,2 P 2,57 2,13 C1orf63: chromosome 1 open reading frame 63 AF247168 84,0 P 216,0 P 2,57 2,22 C15orf39: chromosome 15 open reading frame 39 AL109730 40,6 P 104,0 P 2,56 2,2

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PELO: pelota homolog (Drosophila) NM_015946 372,4 P 955,1 P 2,56 2,4 BCL2L11: BCL2 -like 11 (apoptosis facilitator) AA629050 88,0 P 224,4 P 2,55 2,31 HSPB8: heat shock 22kDa protein 8 AF133207 81,1 P 206,9 P 2,55 2,21 LAMP2: lysosomal -associated membrane protein NM_002294 445,3 P 1130,2 P 2,54 2,34 2 GPR107: G protein -coupled receptor 107 NM_020960 56,5 P 143,2 P 2,53 2,2 HIST1H2BF: histone cluster 1, H2bf NM_003522 92,4 P 233,9 P 2,53 2,28 HIST1H3B: histone cluster 1, H3b NM_003537 44,6 P 112,6 P 2,53 2,12 SEL1L: sel -1 suppressor of lin -12 -like (C. elegans) AB020335 89,5 P 226,4 P 2,53 2,15 HIST2H2AA3 /// HIST2H2AA4: histone cluster 2, BC001629 284,0 P 715,3 P 2,52 2,32 H2aa3 /// histone cluster 2, H2aa4 HOXA2: homeobox A2 NM_006735 52,1 P 131,4 P 2,52 2,17 PFKFB2: 6 -phosphofructo -2-ki nase/fructose -2,6 - AB044805 54,1 A 136,4 P 2,52 2,28 biphosphatase 2 HTATIP2: HIV -1 Tat interactive protein 2, 30kDa AF092095 96,6 P 242,3 P 2,51 2,24 NUDT4 /// NUDT4P1: nudix (nucleoside NM_019094 258,5 P 649,2 P 2,51 2,32 diphosphate linked moiety X)-type motif 4 /// nudix (nucleoside diphosphate linked moiety X)- type motif 4 pseudogene 1

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ORF20 lower Empty ORF20 gene Accession Empty MHV-68 fold change bound Call MHV-68 Call of FC HIST1H3D: histone cluster 1, H3d NM_003530 22,8 A 167,1 P 7,3 5,64 HIST1H2BJ: histone cluster 1, H2bj NM_021058 44,5 A 259,4 P 5,8 5,05 HIST2H4A /// HIST2H4B: histone cluster 2, NM_003548 76,6 P 343,8 P 4,5 4,09 H4a HIST1H2AC: histon e cluster 1, H2ac AL353759 101,5 P 339,2 P 3,3 2,91 ZNF767: zinc finger family member 767 NM_024910 140,0 P 454,4 P 3,2 3,05 DDB2: damage -specific DNA binding protein NM_000107 163,2 P 527,4 P 3,2 2,9 2 HIST1H4H: histone cluster 1, H4h NM_003543 35,2 M 112,1 P 3,2 2,76 ITCH: itchy homolog E3 ubiquitin protein AL109923 156,0 P 493,3 P 3,2 2,83 ligase (mouse) RORA: RAR -related orphan receptor A U04897 40,6 P 128,0 P 3,2 2,45 ITCH: itchy homolog E3 ubiquitin protein AB056663 97,9 P 301,7 P 3,1 2,77 ligase (mouse) HIST1H2BG /// HIST1H2BC: histone cluster 1, NM_003526 17,0 A 50,7 P 3,0 2,23 H2bg /// histone cluster 1, H2bc

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STK17B: serine/threonine kinase 17b NM_004226 53,8 P 157,2 P 2,9 2,41 (apoptosis-inducing) RASSF1: Ras association (RalGDS/AF -6) NM_007182 111,8 P 314,1 P 2,8 2,42 domain family 1 GLS: glutaminase AF097493 96,2 P 269,4 P 2,8 2,53 GPATCH2: G patch domain containing 2 NM_018040 157,2 P 439,0 P 2,8 2,49 GDF15: growth differentiation factor 15 BC000529 46,4 A 129,4 P 2,8 2,25 ITCH: itch y homolog E3 ubiquitin protein AB056663 171,2 P 462,4 P 2,7 2,53 ligase (mouse) HCFC2: host cell factor C2 NM_013320 114,6 P 306,6 P 2,7 2,4 UAP1L1: UDP -N-acteylglucosamine AK022632 61,3 P 159,2 P 2,6 2,29 pyrophosphorylase 1-like 1 ATF7IP: activating t ranscription factor 7 NM_018179 207,6 P 537,7 P 2,6 2,44 interacting protein LOC388796: hypothetical LOC388796 AA827892 343,9 P 862,0 P 2,5 2,32 SFPQ: splicing factor proline/glutamine -rich AL558875 284,5 P 714,5 P 2,5 2,28 (polypyrimidine tract binding protein associated) CIDEC: cell death -inducing DFFA -like NM_022094 95,8 A 238,4 P 2,5 2,27 effector c CLEC4A: C -type lectin domain family 4, NM_016184 28,1 A 69,7 P 2,5 2,08

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member A UBE2H: ubiquitin -conjugating enzyme E2H AI829920 205,4 P 509,6 P 2,5 2,29 (UBC8 homolog, yeast) RORA: RAR -related orphan receptor A L14611 51,7 P 127,6 P 2,5 2,13 GADD45A: growth arrest and DNA -damage - NM_001924 384,7 P 938,4 P 2,4 2,16 inducible, alpha SNX16: sorting nexin 16 NM_022133 48,8 P 119,0 P 2,4 2,08 ETNK1: etha nolamine kinase 1 AL137750 153,8 P 370,3 P 2,4 2,28 PSPC1: paraspeckle component 1 AA969958 84,4 P 200,6 P 2,4 1,94 CROCC /// MGC12760 /// LOC729559: ciliary BC006312 285,1 P 674,3 P 2,4 2,24 rootlet coiled-coil, rootletin THSD1 /// THSD1P: thrombospon din, type I, NM_018676 95,2 A 223,1 P 2,4 2,11 domain containing 1 /// BRF2: BRF2, subunit of RNA polymerase III NM_018310 166,1 P 390,4 P 2,4 2,08 transcription initiation factor, BRF1-like ALB: albumin AF116645 10,1 A 23,8 P 2,4 1,65 RIOK3: RIO kinas e 3 (yeast) /// RIO kinase 3 AW006290 243,5 P 570,8 P 2,3 2,18 (yeast) HIST1H3D: histone cluster 1, H3d NM_021065 55,6 A 129,5 P 2,3 2,1 ARHGEF12: Rho guanine nucleotide AI807672 83,8 A 194,2 P 2,3 1,95 exchange factor (GEF) 12

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ZFX /// ZFY: zinc finger protein, X -linked /// NM_003411 94,0 P 217,5 P 2,3 2,13 ZP3 /// POMZP3 /// MEIS3: zona pellucida NM_012230 58,1 A 134,3 P 2,3 1,99 glycoprotein 3 (sperm receptor) LOC442257: similar to 40S ribosomal protein AL035603 19,6 A 45,3 P 2,3 1,67 S4 IDS: idur onate 2 -sulfatase (Hunter BC006170 9,4 A 21,7 P 2,3 1,55 syndrome) C21orf91: chromosome 21 open reading NM_017447 137,5 P 316,3 P 2,3 2,12 frame 91 KLF6: Kruppel -like factor 6 AB017493 99,6 P 228,6 P 2,3 2,04 TFEC: transcription factor EC NM_012252 11,4 A 26,3 P 2,3 1,67 ATF3: activating transcription factor 3 NM_001674 306,8 P 695,1 P 2,3 2,09 ZNF586: zinc finger protein 586 NM_017652 97,8 P 220,2 P 2,3 2 C6orf48: chromosome 6 ORF 48 NM_016947 675,5 P 1512,7 P 2,2 2,15 LONRF3: LON peptidase N -termin al domain NM_024778 155,9 P 346,6 P 2,2 1,91 and ring finger 3 FGFR1OP /// C9orf4: FGFR1 oncogene NM_007045 300,6 P 658,8 P 2,2 2,07 partner PMS2L11: postmeiotic segregation U38980 343,7 P 752,0 P 2,2 2 increased 2-like 11 ZNF654: zinc finger protein 6 54 NM_018293 53,1 P 116,2 P 2,2 1,98

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CDKN1A: cyclin -dependent kinase inhibitor NM_000389 258,0 P 564,8 P 2,2 1,91 1A (p21, Cip1) ISG20L1: interferon stimulated exonuclease NM_022767 705,4 P 1540,3 P 2,2 2,11 gene 20kDa-like 1 PRKAB2: protein kinase, AMP -activated, NM_005399 86,5 P 188,9 P 2,2 1,99 beta 2 non-catalytic subunit APPBP2: amyloid beta precursor protein NM_006380 204,7 P 444,5 P 2,2 1,97 (cytoplasmic tail) binding protein 2 KHDRBS1: KH domain containing, RNA AW592227 95,3 P 207,2 P 2,2 1,83 binding, signal transduction associated 1 ZNF79: zinc finger protein 79 AA284829 68,6 P 148,4 P 2,2 1,93 TRAPPC2 /// SEDLP: trafficking protein AF291676 220,0 P 473,4 P 2,2 1,98 particle complex 2 /// spondyloepiphyseal dysplasia, late, pseudogene GEM: GTP binding protein overexpressed in NM_00526 61,2 P 131,5 P 2,2 1,94 skeletal muscle 1 UIMC1: ubiquitin interaction motif containing NM_016290 417,2 P 891,4 P 2,1 1,95 1 KLHL24: kelch -like 24 (Drosophila) AW006750 28,0 P 59,6 P 2,1 1,85 HIST1H2AG: histo ne cluster 1, H2ag NM_021064 40,5 A 86,3 P 2,1 1,74 SFRS7: splicing facto r, arginine/serine -rich 7 AA524053 539,0 P 1143,4 P 2,1 1,98

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WEE1: WEE1 homolog (S. pombe) AJ277546 191,3 P 406,1 P 2,1 1,92 KIAA0999: KIAA0999 protein AA044154 113,3 P 240,1 P 2,1 1,9 IFNA5: interferon, alpha 5 NM_002169 13,0 A 27,5 P 2,1 1,58 CCNB1IP1: cyclin B1 interacting protein 1 NM_021178 659,9 P 1382,7 P 2,1 1,94 DKFZp762E1312: hypothetical protein NM_018410 503,4 P 1057,2 P 2,1 1,94 KIF18A: kinesin family member 18A // / NM_031217 227,6 P 478,5 P 2,1 1,88 PMS2L3: postmeiotic segregation increased NM_005395 283,7 P 592,3 P 2,1 1,96 2-like 3 HUS1: HUS1 checkpoint homolog (S. pombe) NM_004507 96,6 P 202,2 P 2,1 1,95 DGKE: diacylglycerol kinase, epsilon 64kDa NM_003647 34,8 P 72,7 P 2,1 1,75 TXNIP: thioredoxin interacting protein NM_006472 133,2 P 277,5 P 2,1 1,89 ZNF79: zinc finger protein 79 X65232 115,8 P 240,7 P 2,1 1,86 ABI3BP: ABI gene family, member 3 (NESH) NM_024801 19,3 A 40,1 P 2,1 1,64 binding protein MSX2 : msh homeobox 2 D31771 257,2 P 531,7 P 2,1 1,85 KLHL24: kelch -like 24 (Drosophila) AW006750 65,1 P 135,1 P 2,1 1,59 CDC27: cell division cycle 27 homolog (S. NM_001256 505,6 P 1039,9 P 2,1 1,97 cerevisiae) C16orf68: chromosome 16 open reading NM_024109 199,5 P 410,4 P 2,1 1,92 frame 68 CCND2: cyclin D2 AW026491 303,4 P 620,6 P 2,1 1,85

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ZNF211: zinc finger protein 211 NM_006385 94,7 P 194,5 P 2,1 1,82 BTG2: BTG family, member 2 BG339064 236,9 P 483,3 P 2,0 1,95 LIF: leukemia inhibitory factor (cholin ergic NM_002309 57,4 A 117,2 P 2,0 1,67 differentiation factor) ARIH1: ariadne homolog, ubiquitin - NM_005744 494,0 P 1001,9 P 2,0 1,88 conjugating enzyme E2 binding protein, 1 (Drosophila) SEC24A: SEC24 related gene family, member BE645231 144,6 P 293,4 P 2,0 1,87 A (S. cerevisiae) ZFX: zinc finger protein, X -linked NM_003410 201,4 P 409,9 P 2,0 1,85 PTEN: phosphatase and tensin homolog U96180 285,1 P 576,0 P 2,0 1,91 (mutated in multiple advanced cancers 1) JOSD3: Josephin domain containing 3 BC001972 642,3 P 1297,7 P 2,0 1,9 NR2C2: nuclear receptor subfamily 2, group NM_003298 70,9 P 143,5 P 2,0 1,78 C, member 2 SEC31A: SEC31 homolog A (S. cerevisiae) U92014 231,1 P 466,4 P 2,0 1,77 PPM1D: protein phosphatase 1D NM_003620 524,3 P 1055,7 P 2,0 1,89 magnesium-dependent, delta isoform HIST2H2AA3 /// HIST2H2AA4: histone cluster BC001629 284,0 P 572,3 P 2,0 1,85 2, H2aa3 /// histone cluster 2, H2aa4 INTS6: integrator complex subunit 6 AL117626 77,8 P 156,6 P 2,0 1,79

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ADRBK2: adrenergic, beta, receptor kinase 2 AI478542 126,2 A 253,6 P 2,0 1,75 ZNF222: zinc finger protein 222 NM_013360 75,5 P 151,8 P 2,0 1,74 INSIG1: insulin induced gene 1 BE300521 347,7 P 696,0 P 2,0 1,9 BRAF: v -raf murine sarcoma viral oncogene NM_004333 91,3 P 182,8 P 2,0 1,82 homolog B1 CDC27: cell division cycle 27 homolog (S. NM_001256 374,1 P 742,7 P 2,0 1,82 cerevisiae) LETMD1: LETM1 domain containing 1 NM_015416 336,3 P 665,5 P 2,0 1,87 LAT1 -3TM /// IMAA /// LOC440345 /// PI -3- NM_031211 261,0 P 517,9 P 2,0 1,82 kinase-related kinase SMG-1 LPP: LIM domain containing preferred AL044018 36,9 A 73,0 P 2,0 1,65 translocation partner in lipoma LRIG2: leucine -rich repeats and NM_014813 103,5 P 202,4 P 2,0 1,72 immunoglobulin-like domains 2 POMZP3: POM (POM121 homolog, rat) and BC000487 56,2 P 110,2 P 2,0 1,65 ZP3 fusion CCNL1: cyclin L1 NM_020307 351,1 P 683,9 P 2,0 1,83 XPO4: exportin 4 NM_022459 110,0 P 214,8 P 2,0 1,79 PDE4DIP /// LOC727927: phosphodiesterase AK001619 56,1 A 109,7 P 2,0 1,64 4D interacting protein (myomegalin) ///

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