MIAMI UNIVERSITY The Graduate School
Certificate for Approving the Dissertation
We hereby approve the Dissertation
of
Anand Prakash
Candidate for the Degree:
Doctor of Philosophy
Dr. Eileen Bridge, Mentor
Dr. Gary R. Janssen, Reader
Dr. Joseph M. Carlin, Reader
Dr. Xiao-Wen Cheng
Dr. David G. Pennock
Graduate School Representative
ABSTRACT
INVESTIGATING THE TRIGGERS FOR ACTIVATING THE CELLULAR DNA DAMAGE RESPONSE DURING ADENOVIRUS INFECTION
by Anand Prakash
Cellular genomic integrity is constantly attacked by a variety of exogenous and endogenous agents. In response to damaged DNA, the cell activates a DNA damage response (DDR) pathway to maintain genomic integrity. Cells can also activate DDRs in response to infection with several types of viruses. The cellular DDR pathway involves sensing DNA damage by the Mre11, Rad50, Nbs1 (MRN) sensor complex, which activates downstream ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) kinases. These kinases phosphorylate downstream effector proteins implicated in cell cycle arrest, DNA repair, and, if the damage is irreparable, apoptosis. The induction of DDRs includes focal accumulation and phosphorylation of several DDR proteins. Adenovirus (Ad) mutants that lack early region 4 (E4) activate a cellular DDR. E4 proteins normally inactivate the MRN sensor complex and prevent downstream DDR signaling involved in DNA repair and cell cycle checkpoint arrest in wild- type Ad5 infections. The characteristics of Ad infection that activate the cellular DDR are not well understood. We have investigated the ability of replication defective and replication competent Ad mutants to activate cellular DDRs and G2/M cell cycle arrest. Ad infection induced early focal accumulation of DDR proteins such as Mre11, Mdc1, phosphorylated ATM (pATM), phosphorylated Chk2 (pChk2), and 53BPI, independent of the replication status of the mutants studied. However, Mre11 and pATM foci were transient in replication defective infections and were only maintained in infections with replication competent mutants. Viral DNA replication was correlated with amplification of pATM levels as well as its substrates, pChk2 and pNbs1. Furthermore, we found that G2/M cell cycle arrest was not activated by a replication defective mutant or a mutant expressing the E4orf3 encoded 11kDa protein. Our results suggest that the initial induction of DDR foci does not require viral DNA replication. In contrast, viral DNA replication is important for maintenance of DDR proteins at viral replication centers, amplification of pATM, pChk2, and pNbs1, and G2/M cell cycle arrest.
INVESTIGATING THE TRIGGERS FOR ACTIVATING THE CELLULAR DNA DAMAGE RESPONSE DURING ADENOVIRUS INFECTION
A DISSERTATION
Submitted to the Faculty of
Miami University in partial
fulfillment of the requirements
for the degree of
Doctor of Philosophy
Department of Microbiology
by
Anand Prakash Miami University Oxford, Ohio 2014
Dissertation Director: Eileen Bridge, Ph.D.
Table of Contents
List of Tables iii
List of Figures iv
Acknowledgements vi
General Introduction 1
Chapter I. Differential activation of cellular DNA damage responses by 16 replication defective and replication competent adenovirus mutants
Chapter II. Adenovirus DNA replication amplifies ATM dependent 56 signaling and contributes to the induction G2/M cell cycle arrest during activation of the DNA damage response
Concluding Remarks 81
References 92
ii
List of Tables
Table 1. Status of E4-11kDa and E4-34kDa gene in the E4 22 mutants used in this study.
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List of Figures
Figure 1. Diagrammatic representation of Adenovirus genome. 3
Figure 2. Model of Adenovirus DNA replication. 6
Figure 3. Cellular DNA Damage Response (DDR) activation. 10
Figure 4. Mdc1 focus formation in Ad-infected cells is correlated 27 with MOI.
Figure 5. AdRSVβgal is defective for viral early gene expression 30 and viral DNA replication.
Figure 6. Mdc1 focus formation depends on transcription from the 33 viral genome.
Figure 7. Replication defective AdRSVβgal fails to activate phosphorylation 36 of Nbs1 and Chk1.
Figure 8. Redistribution of Mre11 to nuclear tracks is not sufficient to prevent 39
Nbs1 phosphorylation.
Figure 9. UV treatment of AdRSVβgal infected cells activates phosphorylation 42 of Nbs1.
Figure 10. Viral DNA replication is important for activating Nbs1 phosphorylation. 45
Figure 11. E4 mutant-induced phosphorylation of Nbs1 and Chk1 correlates with the 48 onset of viral DNA replication.
Figure 12. Focal accumulation of pATM is not maintained in the absence of DNA 66 replication or in the presence of E4-11kDa.
Figure 13. ATM substrates are detected in foci in the absence of viral DNA 69 replication but an increase in phosphoprotein levels is not detected in western blot analysis.
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Figure 14. Viral DNA replication amplifies ATM dependent signaling. 72
Figure 15. G2/M cell cycle arrest occurs in 1007 but not in 1010 or E1- infections 75
Figure 16. Model for Ad induced cellular DDR. 89
v
Acknowledgements
I am grateful to my advisor Dr. Eileen Bridge for her guidance, support, and constant encouragement during my PhD carrier. I thank you for sincerely taking time out from your busy schedule to teach me to think in scientific manner. I deeply appreciated you correcting endless drafts of non-sequitur writing and guiding me to improve my scientific and thesis writing. You have been caring and supportive mentor in the matters non-scientific as well. I will miss talking to you about your animals.
I thank my doctoral committee members who were instrument in my scientific growth. Dr Gary Janssen, thank you for your generous critiques on my manuscript and my dissertation. I appreciate you for your advice during the job negotiation, words of encouragement during my PhD, and philosophical conversations about the comic books. Dr. Joseph Carlin, thanks for reading and providing your valuable critiques on my dissertation. Also, your questions and comments during the various journal clubs, I have attended, enriched me scientifically. Dr. Xiao- Wen Cheng, thank you for providing guidance, suggestions, reagents, and access to your lab and office at any time. Dr. David Pennock, thank you for providing my valuable suggestions during my committee meetings. Thanks for being very accommodative.
I thank people in the department of Microbiology who had helped me consciously and unconsciously over the years. Dipendra Gautam – I couldn’t ask for a better friend. Your memories have become inseparable from Oxford memories. You have taught me a lot and looking back, I see, I haven’t thanked you enough. Thank you! Jenna Dolhi –I don’t remember a single instance when I needed your help and you said no. Thanks for listening, helping, and being such a beautiful soul. Rachael Desmone – thoughtful, considerate, caring, and most down to earth I have met in USA. Thanks. Chris Sedlacek – an Indian friend outside of India. Thanks for all those conversations and dinners and making me feel home. Tyler Garretson and Nicole Marotta – you are rockstar TAs and wonderful human beings. Akhilesh Kumar, Ashwani Kumar, Subash Dhungana, and Amber Beckett – interacting with you people have always been blast. I wish you all the best in whatever journey you take. Barb and Darlene, thank you for your unconditional help! Like goddess, you have powers of being omniscient and you know exactly what needs to be done under a given circumstances, however grave that might sound. I am grateful to everyone in the department for suggestions, advices, helps, reagents, and laughs. vi
Vishal Kumar (Zoology) and Faizule Hasan Lincoln (Biochemistry), thanks for all the jokes, foods, and movie talks. I will miss you guys.
I want to thank all the baristas and people at Kofenya (for I have spent half of my grad life there) for welcoming me with open arms and serving me with much needed caffeine and laughter. Following people need special mention apart from caffeine atmosphere: Derek Reeverts (for deep conversation and advice on writing, myths, psychology, and personal matters), Joel Griggs (for sharing music and talks about pop culture), Brady Turner (Grant Morrison Fandom), Kathryn Marsman (telling me to read: The Lion, the Witch and the Wardrobe, apart from being so graceful), Bethany Bateman and Nathan Holstein (you are synonymous with great pancakes and robust coffee), Spencer Birchfield (for all those conversations, breakfasts, and jokes), Liz Snyder (Nothing would have been possible without you setting up this great atmosphere), Erin Sams, Sevika Balachandra, and Cecilia Stelzer (for reading and commenting on my stories). All of you will stay in my heart and in my memories. All of you will probably appear in my writings in one form or another.
For keeping me sane throughout PhD, I owe a great deal to my friends: Yadvinder Singh (for all those philosophical discussion about Mahabharata and life), Yogi Singh Rajawat (for telling me things I should be doing like a big brother), Sandeep Kumar (just thinking about you makes me happy), Ram Prakash Kamal (for understanding me), Amandeep Dhaliwal (for laughing at my jokes), Kavita (for constantly asking about my wellbeing) and Karuna (for reminding me about my roots: Haryana).
My dear family for your love and support in everything I wanted to do in life. Mummy and Papa for all the sacrifices you have made for your children and making me who I am. Arti and Sarita, my lovely sisters, for loving, fighting, and arguing with me, sharing details about the adventures for growing Arpita, Riya, and Chetan on weekly basis. Chand Singh and Dheeraj Chauhan, my elder and younger brothers, former for teaching me things, I probably don’t have memories of, and latter for being my quintessential ‘filmy’ younger brother who thinks world should understand him. Roshan Singh and Surinder Kumar, my brother-in-laws, for all the happiness you brought to me and my family. Satbir Kajal, my brother, for being there for me every single time and my buwa for her unconditional affection. Thank you everyone.
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General Introduction
Viruses encounter many challenges inside the host cell that impact their survival and successful progeny production. The virus must take advantage of cellular machinery to complete its life cycle while the cell establishes many barriers to virus propagation. The cellular DNA damage response (DDR) can present either an opportunity or a barrier to a productive virus infection. Viral genomes, viral protein production, and viral genome replication, alone or in combination, can potentially induce a cellular DDR. Many DNA and RNA viruses manipulate the cellular DDR for successful replication, progeny production, and in some cases, establishment of latency (reviewed in McLaughlin-Drubin and Munger, 2008). Human Papillomavirus (HPV) and Epstein-Barr virus (EBV) activate the cellular DDR for their own benefit. In contrast, Adenovirus (Ad) type 5 must inactivate the cellular DDR to create a cellular environment amenable to viral DNA replication and progeny production. Early region 4 (E4) proteins inactivate the DDR, and consequently an Ad mutant in which E4 is deleted induces a cellular DDR. Although the linear viral dsDNA genome may mimic double strand breaks (DSBs) in a host chromosome, the characteristics of Ad infection that are required to activate a cellular DDR are not well understood. Ad-induced cellular DDR regulates downstream cellular processes such as cell cycle arrest and DNA repair to form viral genome concatemers. In this study, we use several Ad mutants to investigate activation triggers for inducing cellular DDRs, and investigate the impact of Ad-induced cellular DDRs on cell cycle arrest.
Historical Background
Ad belongs to the family Adenoviridae which consists of a large number of DNA viruses known to infect mammals and birds. In humans, Ad infection is usually associated with mild respiratory illness, conjunctivitis, and gastroenteritis but it also causes debilitating inflammatory conditions in immunocompromised individuals (reviewed in Echavarria, 2008). Ad has been used as a tool to understand various cellular processes such as messenger RNA splicing and alternative polyadenylation (Berget et al., 1977, Skolnik-David et al., 1987). Though Ad does not cause cancer in humans, it can transform mammalian cell lines and causes tumors in rodent cells (Graham et al., 1997). The transformation and tumorigenic potential of Ad is mediated by early viral proteins which inactivate tumor suppressors such as pRb and p53 (reviewed in Xiao et al.,
1
2005). Also, functional understanding of Ad viral proteins has been instrumental in developing viral vectors for the purpose of gene therapy and tumor cell killing (Weitzman, 2005). Thus, Ad serves as a model system to understand host cell processes, virus-host interactions, carcinogenesis, and development of viral vectors.
Morphology and genome organization
Ad is a non-enveloped virus which encases its linear double stranded (ds) DNA in an icosahedral capsid. The capsid is composed of the major viral structural proteins, hexon, penton, and fiber. These proteins are important in recognition and entry of host cells. The viral genome is approximately 36kbp in length and has terminal proteins covalently attached to its 5’ ends. The ends of the viral genome have ~ 100 base pair long inverted terminal repeats (ITRs) which contain the viral origin of replication (Shenk, 1996). Both ITRs and terminal proteins are involved in viral DNA replication. The transcription of early genes precedes viral DNA replication, which is followed by late gene transcription. The Ad genome is transcribed in a temporal manner, where early genes E1a, E1b, E2, E3, and E4 give rise to early transcripts and the late genes are subsequently transcribed from the major late transcription unit (MLTU). Viral transcripts undergo alternative splicing and polyadenylation to give rise to multiple mRNAs for protein production (Fig. 1) (reviewed in Russell, 2000). Viral proteins regulate viral gene expression and cellular pathways involved in controlling the cell cycle, apoptosis, and the DDR, to create a cellular environment amenable to viral replication and progeny production (reviewed in Weitzman and Ornelles, 2005). The viral early proteins are required for viral DNA replication, which serves as a switch for activating late transcription. The late transcripts encode structural proteins important for assembling virus particles.
Life cycle of Adenovirus
The Ad life cycle begins with the interaction of viral capsid proteins with host cell receptors. First, fiber proteins of the viral capsid interact with the Coxsackie-Adenovirus Receptor (CAR) on host cells. This interaction is further reinforced by binding of penton proteins with cellular integrin receptors, αvβ3 or αvβ5 (reviewed in Echavarria, 2008). Upon binding to
2
Figure 1. Diagrammatic representation of Adenovirus genome. The linear ds DNA of the Ad genome is linked to 5’ terminal proteins (TP) on either side. Early genes are shown in red and late genes expressed from the major late major late transcription unit (MLTU) are shown in dark blue. Arrows indicate the transcription start site and the direction of transcription. Two major E4 encoded proteins, E4 11kDa and E4 34kDa, are shown here.
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cellular receptors, the virus is internalized by clathrin-mediated endocytosis (reviewed in Meier and Greber, 2004). Endosomal acidification causes conformational changes in the viral penton protein and the partially uncoated virus is released into the cytosol. The virus is transported from the cytosol to the nuclear pore complex via microtubules. The viral DNA remains associated with core associated proteins, protein V, VI, VII, mu, and covalently bound terminal proteins when it reaches the nucleus (Anderson et al., 1989; Matthews and Russell, 1995).
Host RNA polymerase II initiates transcription of the viral E1a gene, which encodes the E1a protein. E1a is a major transcription activator that regulates transcription of other early viral genes important in viral replication. E1a also pushes the host cells into S phase to promote efficient viral DNA replication. The E1b encoded proteins inhibit apoptosis, selectively export late Ad mRNA into cytoplasm, and inactivate cellular DDR proteins (reviewed in Berk, 2005). The E1 region encodes for multifunctional proteins, and Ad E1 mutants are defective for early gene expression and viral DNA replication (Berk et al., 1979). Viral DNA replication requires the combined action of E2 gene products. The E3 proteins are important in immune evasion and the spread of virus in infected animals (Lichtenstein et al., 2004). However, loss of the E3 genetic region does not interfere with virus growth in cell culture models. The E4 gene products significantly affect a number of viral and cellular processes such as transcription, viral DNA replication, RNA splicing, late protein synthesis, and the cellular DDR response (Weitzman, 2005).
Ad DNA replication requires viral DNA polymerase and pre-terminal protein (pTP). Ad replicates its DNA in two phases using a unique protein priming mechanism. In the first phase, pTP serves as a protein primer for the viral DNA polymerase, which synthesizes a new viral strand complementary to the template strand. In this process, one of the parental DNA strands is displaced. The complementary ITRs of the displaced ss DNA strand anneal and give rise to a ‘pan-handle’ structure with a double stranded handle. In the second phase, pTP and viral DNA polymerase bind to the ds ITRs of the pan-handle and initiate replication, similar to first phase. Thus, Ad genome replication gives rise to single stranded (ss) DNA intermediates, ds DNA, and unusual replication intermediates such as the pan-handle structure (Fig. 2) (Challberg and Kelly, 1989).
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Figure 2: Model of Adenovirus DNA replication. DNA replication initiates from the end of the genome and displaces the non-template strand. The displaced strand forms a pan handle by self- base pairing at inverted terminal repeats. DNA replication initiates at the handle and forms a double strand. This two-step replication process yields two semi-conserved genomic DNAs (Fig. from Gautam D. (2013) PhD thesis)
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The onset of viral DNA replication increases transcription from the MLTU. A large transcript is synthesized, which undergoes alternative splicing and polyadenylation to give rise to multiple late mRNAs. The late mRNAs encode structural proteins required to package the viral genome into virus particles. Virus particles are released from the host cell following cell lysis.
Overview of the cellular DNA damage response (DDR)
Maintaining genomic integrity holds paramount importance for the cell in terms of its survival and successful reproduction. The cellular genome is constantly attacked by exogenous agents including ionizing radiation (IR), ultraviolet (UV) radiation, and radiomimetic drugs as well as endogenous agents such as metabolic reactive oxygen free radicals, meiotic recombination, and replication fork collapse. The cell activates a DDR in response to these damaging agents. This is a multistep process that includes sensing the DNA damage, activating downstream kinases and mediator proteins, and transducing the signals to effector proteins. The effector proteins determine the fate of the damaged cell. The cell can be halted at cell cycle checkpoints to repair the damaged DNA, or if the DNA damage is too severe to repair, can undergo apoptosis. Overall, the cell activates DDRs to prevent the transfer of damaged DNA to daughter cells and maintain genome integrity (Weitzman et al., 2010).
DNA damage is sensed by the Mre11/ Rad50/ Nbs1 (MRN) complex, which activates downstream phosphatidylinositidol-3-kinase (PI-3) protein kinases such as Ataxia-telangiectasia mutated (ATM), and ATM and Rad3 related (ATR). ATM mainly responds to DSBs in the genome induced by IR and radiomimetic drugs, while ATR is primarily activated in response to ss DNA generated by stalled replication forks and UV treatment (Bakkenist and Kastan, 2004; Zou and Elledge, 2000) (Fig. 3).
IR and radiomimetic drugs induce DSBs. DSBs are a very detrimental type of DNA damage as these involve loss of genomic regions, genomic rearrangements and may lead to cell death (Derheimer and Kastan, 2010). In response to DSBs, the MRN sensor complex recruits ATM to the site of damage (Falck et al., 2005). The activation of ATM involves autophosophorylation and subsequent monomerization (Bakkenist and Kastan, 2003). Activated ATM phosphorylates histone variant H2AX (γ-H2AX) in the proximity of the DSBs (reviewed in Huen and Chen, 2007) and extends γ-H2AX signals megabase pairs away from the DSB 8
(Burma et al., 2001; Rogakou et al., 1999). The γ-H2AX protein serves as docking site for mediator of DNA damage checkpoint protein 1 (Mdc1) (Lukas et al., 2004). Mdc1 is a critical DDR amplifier as it recruits multiple DDR proteins. Mdc1 recruits additional phosphorylated ATM (pATM) to DSBs (Chapman and Jackson, 2008; So et al., 2009), which will phosphorylate more H2AX molecules, which in turn recruits more Mdc1, thereby amplifying the cellular DDR. The pATM kinase also phosphorylates Mdc1 to recruit E3 ubiquitin ligases, RNF8 and RNF168, which ubiquitinate H2A and H2AX, which in turn serve as binding sites for BRCA1 and 53BPI (reviewed in Huen and Chen, 2007). Thus, IR induces accumulation of multiple DDR proteins at DSBs.
The accumulation of DDR proteins at DSBs following IR can be visualized as microscopic foci and are defined as Ionizing Radiation Induced Foci (IRIF). In IRIF, DDR proteins are recruited to the site of DNA damage in a temporal manner and interact via protein- protein interactions (Nagy and Soutoglou, 2009; Polo and Jackson, 2011). The DDR proteins at the site of DNA damage are also subjected to various types of post-translational modifications (PTMs), which include phosphorylation, ubiquitylation, and sumolyation, all of which help to regulate the DDR. The function of IRIF are not completely understood, but they have been implicated in cell cycle arrest and DNA repair (Coster and Goldberg, 2010; Huen and Chen, 2007).
Cell cycle progression can be arrested at different checkpoints to assess the cell state prior to entering the next phase of the cell cycle. This ensures correct cell division and transfer of cell contents and proper genetic material to daughter cell. The cell cycle can be blocked at G1, S, or G2/M checkpoints. Cyclins and cyclin dependent kinases (Cdks) play important roles in regulation of different checkpoints. The cellular transition into mitosis requires the activity of cyclin dependent protein kinase, Cdc2, which is phosphorylated during the S and G2 phase of the cell cycle. The Cdc25c phosphatase dephosphorylates Cdc2 and pushes the cell into mitosis (Graves et al., 2001; Abraham, 2001). During the DDR, ATM and the functionally related kinase ATR, phosphorylate Chk2 and Chk1, respectively. The phosphorylated Chk1 (pChk1) and phosphorylated Chk2 (pChk2) inactivate Cdc25c by phosphorylation at serine-216 (Matsuoka et al., 1998). The inactivated Cdc25c is not able to dephosphorylate Cdc2, and since this is necessary for progression to mitosis, the cell cycle is arrested at G2/M (Peng et al., 1997; 9
Figure 3: Cellular DNA Damage Response (DDR) activation. Exogenous and endogenous factors induce damage to cellular DNA. The damage is detected by a sensor protein complex (e.g., MRN) which sends signals to the downstream kinases ATM and/or ATR. ATM primarily responds to DSBs. ssDNA is a major trigger for the ATR response. Activation of these kinases transduces signals to downstream effector proteins involved in DNA repair, cell cycle arrest, and apoptosis.
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Bahassi et al., 2006). Thus, during the DDR, kinases inactivate the Cdc25 phosphatase which regulates Cdc2 to arrest the cell cycle at the G2/M checkpoint.
Cells repair damaged DNA by two primary mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR uses the homologous sister strand for accurate repair whereas NHEJ is a quick, error prone pathway that ligates broken DNA ends together without using homologous sequences. The choice of DNA repair pathway is dependent upon cell cycle phases. HR repair primarily takes place during S and G2 phases of the cell cycle when the homologous sister strand is available. NHEJ repairs damaged DNA during any phase of cell cycle (Mao et al., 2008). During NHEJ, a heterodimer of the Ku70/Ku80 proteins binds to the ends of a DSB and recruits DNA-PK. The broken DNA ends are then processed by various nucleases and polymerases. Finally, DNA ligase IV and its co-factors XRCC4 and XLF repair the processed ends by ligation (Weterings and Chen, 2008).
Proper regulation of cell cycle checkpoint arrest allows time for DNA repair, complete DNA replication, successful transition between different cell cycle phases, and proper segregation of genetic material to daughter cells. In order to maintain genomic integrity, the cell must successfully repair damaged DNA or undergo apoptosis if the DNA damage is irreparable. Any defects in cellular DDRs may lead to genomic instability, cellular transformation, and tumorigenesis.
Role of Adenovirus E4 gene products in DDR inactivation
The E4 promoter in wild type Ad5 generates a single primary transcript which undergoes alternate splicing to give rise to 18 distinct mRNA. Six different E4 proteins are demonstrated in infected cells (reviewed in Weitzman, 2005). Many of the E4 proteins are multifunctional in nature and deletion of the entire E4 region results in defective viral DNA replication, reduced late gene expression, formation of viral genome concatamers, and reduced viral yield (Halbert et al., 1985; Weinberg and Ketner, 1986; Bridge and Ketner, 1989; Huang and Hearing, 1989; Weiden and Ginsberg, 1994). E4 produces an 11kDa protein from E4orf3 (E4 11kDa) and a 34kDa protein from E4orf6 (E4 34kDa) and both these proteins have shown functional redundancy in regulating viral DNA replication, late protein synthesis and progeny production (Bridge and Ketner, 1989; Huang and Hearing, 1989). 12
E4-11kDa and E4-34kDa inactivate multiple cellular DDR proteins in wild type Ad5 infections. E4 11kDa inactivates many tumor suppressor proteins such as promyelocytic leukemia protein (PML), p53, TRIM24, and the MRN complex by redistributing them into track like structures (Doucas et al., 1996; Stracker et al., 2002; Yondola and Hearing, 2007). E4- 11kDa also inactivates Mre11 by redistributing it to cytoplasmic aggresomes, which are sites for accelerated proteasomal degradation (Araujo et al., 2005; Liu et al., 2005). Furthermore, E4- 11kDa inactivates p53 by a unique mechanism where E4-11kDa directs a cellular methyltransferase to selectively induce repressive heterochromatin marks at p53 dependent promoters, thereby inhibiting p53 binding (Soria et al., 2010). E4-34kDa also inactivates cellular DDR proteins. The E4 34kDa protein makes a complex with E1b 55kDa and this complex binds an E3 ubiquitin ligase which ubiquitinates a number of cellular DDR proteins and targets them for proteasomal degradation (Harada et al., 2002). The proteins which are degraded by the E4 34kDa - E1b 55kDa complex include Mre11, p53, DNA ligase IV, BLM, Tip60, and Daxx (Stracker et al., 2002; Querido et al., 2001; Baker et al., 2007; Orazio et al., 2011; Gupta et al., 2012; Schreiner et al., 2013). Thus, E4- 11kDa and E4-34kDa inactivate multiple cellular DDR proteins; in the absence of these viral proteins, the MRN sensor complex activates downstream ATM and ATR dependent cellular DDRs (Stracker et al., 2002; Carson et al., 2003; Carson et al., 2009).
E4-11kDa and E4-34kDa regulate DDR signaling, cell cycle arrest, and viral DNA repair. E4- 11kDa-mediated Mre11 redistribution is sufficient to abrogate ATR signaling, whereas Mre11 degradation by E4 34kDa inactivates both ATM and ATR signaling (Carson et al., 2003; Carson et al., 2009). E4 mutants also induce G2/M cell cycle arrest (Cherubini et al., 2006) which could be the result of ATM and/or ATR activation. The E4-11kDa and E4-34kDa proteins physically associate with and interfere with the activity of DNA-PK, which is a critical kinase for NHEJ repair and viral genome concatenation (Boyer et al., 1999). The E4-34kDa - E1b 55kDa complex degrades DNA ligase IV, which is responsible for ligating DNA ends together during the last step in NHEJ repair (Baker et al., 2007). Thus, in the presence of E4 proteins, the NHEJ repair pathway is inactivated and wild type Ad5 exists as linear ds DNA genome monomer. In contrast, E4 deleted mutants exhibit viral genome concatenation in which end-to-end linked viral genomes are present as multimers (Weiden and Ginsberg, 1994). Taken together, E4 proteins
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inactivate ATM and ATR dependent signaling, prevent G2/M arrest, and abolish viral genome concatenation during Ad infections.
Activation triggers of the cellular DNA damage response in Adenovirus infection
The characteristics of Ad infection required to induce the cellular DDR are not well understood. Ad infection introduces linear ds DNA to the nucleus. The incoming linear ds DNA with covalently attached terminal proteins and associated core-binding proteins V and VII could be sufficient to activate a cellular DDR. Expression of the E1a protein pushes the cell into S phase and this may potentially activate DDRs (Cuconati et al., 2003). Ad replication generates multiple copies of linear ss DNA, ds DNA, and unusual replication intermediates such as pan handle structures, all of which could act as triggers for the cellular DDR. Thus, different steps in viral life cycle are potential DDR activation triggers. The cellular DDR induced by Ad infection includes focal accumulation and phosphorylation of multiple DDR proteins. We have observed focal accumulation of the DDR protein, Mdc1, in both wild type Ad5 and E4 mutant infections as early as 4 hours post infection (hpi), before the onset of viral DNA replication, which occurs at 10-12 hpi (Mathew and Bridge, 2007). Infection with E2-deleted replication defective Ad induces γH2AX foci formation (Nichols et al., 2009). Karen et al. (2011) have shown that phosphorylated ATM (pATM) foci are observed in an E4 mutant before the onset of viral DNA replication. However, the induction of p53 and Chk1 phosphorylation is observed after the onset of viral DNA replication (12 hpi) in time course experiments (Carson et al., 2003). These findings suggest that some DDR proteins are activated in the absence of viral DNA replication while others may be activated upon viral DNA replication. Replication competent E4 mutant infection activates phosphorylation of ATM and its effector substrate Chk2 (Carson et al., 2003) and induces G2/M cell cycle arrest (Cherubini et al., 2006). We used replication defective and replication competent Ad mutants to investigate the possible activation triggers for the cellular DDR during Ad infection and the impact of viral DNA replication on DDR activation and its relationship to G2/M cell cycle arrest.
We find that a replication defective mutant induces Mdc1 foci formation while replication competent E4 mutants phosphorylate Nbs1, suggesting that the incoming viral genome is important for Mdc1 foci formation whereas viral DNA replication is important for
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Nbs1 phosphorylation (Chapter I). Although Ad infection induces focal accumulation of several DDR proteins before viral DNA replication, we observe that viral DNA replication is important for maintenance of DDR proteins at foci, amplifying levels of phosphorylated DDR proteins, and G2/M cell cycle arrest (Chapter II).
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Differential activation of cellular DNA damage responses by replication defective and replication competent adenovirus mutants
Anand Prakasha, Sumithra Jayarama*, and Eileen Bridgea,b
Published in Journal of Virology, 2012. 86(24):13324-13333
.
Department of Microbiologya and Cell, Molecular and Structural Biology Programb
Miami University, Oxford, Ohio, USA
*Current address: 2103 Stearns Hill Road
Waltham MA 02451
Address correspondence to Eileen Bridge, [email protected]
Running Title: Ad activation of DNA damage responses
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Abstract
Adenovirus (Ad) mutants that lack early region 4 (E4) activate phosphorylation of cellular DNA damage response proteins. In wild type adenovirus type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We have investigated activation of DNA damage responses by a replication defective Ad vector (AdRSVgal), which lacks E1 and fails to produce the immediate early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units including E4. AdRSVgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVgal infected cells to induce cellular DNA damage responses. AdRSVgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins.
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Introduction
Ad contains a 36kbp double-stranded linear DNA genome. The protein products of early region (E4) are important for modulating splicing, apoptosis, transcription, DNA replication, and DNA repair pathways (reviewed in Tauber and Dobner, 2001; Weitzman and Ornelles, 2005). Infection with E4 mutants induces a cellular DNA damage response (DDR) that involves activation of DNA repair kinases ataxia telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) (Carson et al., 2003), which are critical for mediating responses to DNA damage. Cells have evolved an elaborate network of sensor, transducer, and effector proteins that coordinate cell-cycle progression with the repair of DNA damage (reviewed in Harper and Elledge, 2007). Autophosphorylation and activation of the ATM kinase is one of the earliest characterized events in response to double strand breaks (DSBs). Autophosphorylation of ATM at serine 1981 leads to dimer dissociation, and it has been proposed that this leads to the release of active ATM monomers that phosphorylate downstream effector molecules such as the protein product of the gene responsible for Nijmegen breakage syndrome (Nbs1), 53BP1, Chk2, histone H2AX, mediator of DNA damage checkpoint protein 1 (Mdc1), and BRCA1 (Bakkenist and Kastan, 2003; Kurz and Less-Miller, 2004). The Mre11/Rad50/Nbs1 (MRN) complex is important for ATM activation and phosphorylation of a number of proteins involved in DNA repair and checkpoint signaling (Lee and Paull, 2005). ATM autophosphorylation and downstream signaling is profoundly impaired in infections with wild-type Adenovirus type 5 (Ad5) due to degradation of MRN complex proteins (Carson et al., 2003), an observation consistent with the idea that the MRN complex functions as a DNA damage sensor that collaborates with transducing kinases to activate DNA repair, cell cycle checkpoint, and apoptosis pathways. The MRN complex also plays an important role in the physical repair of DSBs by providing a scaffold that holds DNA breaks together during ligation and repair (Assenmacher and Hopfner, 2004). Thus, the MRN complex acts as both a sensor and an effector of ATM activation and signaling in response to E4 mutant infections and after the introduction of DNA DSBs (Carson et al., 2003; Lee and Paull, 2005). ATR is also active following E4 mutant infections. ATR responds to several types of DNA damage, but a common theme is the presence of RPA-coated single stranded DNA (ssDNA) that is produced during repair of damaged DNA or when replication forks stall at sites of DNA damage (Cimprich and Cortez, 2008; Zou, 2007). Ad DNA
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replication produces ssDNA intermediates during its replication (van der Vilet, 1995) that could also serve to activate ATR responses.
The cellular DDR induced by E4 mutant infection inhibits viral DNA replication (Evans and Hearing, 2005; Lakdawala et al., 2008; Mathew and Bridge, 2007; Mathew and Bridge, 2008) and results in the concatenation of viral genomes (Boyer et al., 1999; Stracker et al., 2002; Weiden and Ginsberg, 1994). Ad has evolved several mechanisms to counteract the detrimental effects of the DDR on its life cycle. E4 produces an 11-kDa protein from open reading frame (ORF) 3 (E4-11kDa) and 34-kDa protein from ORF6 (E4-34kDa) that each form a physical complex with DNA-PK, which is a critical enzyme for repair by non-homologous end-joining and for the production of E4 mutant genome concatemers (Boyer et al., 1999). E4-34kDa forms a complex with the E1b-55kDa protein and interacts with a cellular CUL5-containing E3 ubiquitin ligase (Blanchette et al., 2004; Ying Cheng et al., 2007; Querido et al., 2001). This complex targets several DDR proteins for ubiquitination and proteasome-mediated degradation, including Mre11 of the MRN complex (Carson et al., 2003; Stracker et al., 2002), ligase IV (Baker et al., 2007), and the cellular tumor suppressor p53 (Querido et al., 2001). E4-11kDa causes redistribution of MRN complex proteins away from sites of active viral DNA replication to nuclear track-like structures (Evans and Hearing, 2005; Stracker et al., 2002), and cytoplasmic aggresomes located at the periphery of the nucleus (Araujo et al., 2005).
The features of Ad infection required to induce the cellular DDR are not yet completely understood. Incoming genomes are linear double-stranded DNA templates with covalently attached 5' terminal proteins, and are associated with the virion core DNA binding proteins V and VII (Sergeant et al., 1979; Vayda et al., 1983). This template could itself serve as a trigger for activating cellular DDRs (Weitzman and Ornelles, 2005). Viral early gene expression produces multiple regulatory proteins. The E1a protein has transforming properties that stimulate cells to enter S phase (Whyte et al., 1989) and could potentially contribute to activation of the cellular DDR (Cuconati et al., 2003). Ad replicates its DNA by a protein priming and strand displacement mechanism, using its own DNA polymerase (Challberg and Kelly, 1989; van der Vilet, 1995). The presence of unusual replication intermediates and ssDNA produced during E4 mutant replication could also potentially activate the DDR.
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Localization of the DDR protein Mdc1 in discrete foci and phosphorylation of the histone variant H2AX are among the earliest events in the DDR to ionizing radiation (reviewed in Coster and Goldberg, 2010). We have observed Mdc1 in foci at early times after infection with either Ad5 or an E4 mutant, prior to the onset of viral DNA replication (Mathew and Bridge, 2007). Nichols et al. (2009), found that a replication defective virus lacking E2 is still able to activate focal accumulations of phosphorylated H2AX (γH2AX), suggesting that some aspects of the DDR do not require viral DNA replication. Karen and Hearing (2011) recently found that transcription-mediated remodeling of the Ad genome (Chen et al., 2007) was critical for activation of ATM phosphorylation, suggesting that the chromatin structure of the incoming viral genome may prevent DDR activation.
We have investigated induction of DDRs by a replication defective Ad vector, AdRSVβgal, which carries the β-galactosidase gene driven by the Rous Sarcoma Virus promoter in place of the viral E1 region (Stratford-Perricaudet et al., 1992). This vector lacks the E1 genes needed to efficiently activate transcription of the other viral early genes, including the E2 genes that encode viral DNA replication proteins, and the E4 genes responsible for inactivating the cellular DDR. This mutant is thus profoundly defective, both for viral early gene expression and DNA replication, although it does synthesize β-galactosidase from the engineered expression cassette. We find that AdRSVβgal is able to activate redistribution of Mdc1 proteins into foci. Mdc1 focus formation correlates with multiplicity of infection (MOI), consistent with the idea that this protein forms foci in response to incoming viral DNA genomes. Interestingly, replication defective AdRSVβgal infection does not result in phosphorylation of Nbs1 and Chk1, which are normally phosphorylated by the kinases ATM and/or ATR during the cellular DDR. We see a strong correlation between the ability to replicate viral DNA and phosphorylation of Nbs1 and Chk1. Our results suggest that incoming viral genomes may be sufficient to stimulate some aspects of the cellular DDR, but other repair responses may be activated by the process of viral DNA replication.
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Materials and Methods
Cells and viruses. HeLa, W162 (Weinberg and Ketner, 1983), and HEK293 cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 10 U/ml penicillin and 10 µg/ml streptomycin. Wild type Ad5 and E4 mutants were propagated on W162 cells, which complement the E4 mutant defect. E4 mutants used in the present study include H5dl1007, H5dl1010, and H5dl1014 (Bridge and Ketner, 1989). The status of the E4 genetic regions encoding E4-11kDa and E4-34kDa in each of the mutants is indicated in Table 1. AdRSVβgal (Stratford-Perricaudet et al., 1992) was propagated on HEK293 cells which supply E1 gene products in trans. Ad5 titers were determined on both HEK293 and W162 cells and expressed as fluorescent focus forming units (FFU)/ml (Phillipson, 1961). The titers for AdRSVβgal and the E4 mutants were determined on HEK293 and W162 cells, respectively. A conversion factor using Ad5 titers on both HEK293 and W162 cells was calculated, and was used to determine the W162 titer equivalent for AdRSVβgal. HeLa cells were infected at different MOIs using W162 titers of Ad5 and the mutants. In some experiments, virion DNA was prepared from aliquots of purified virus stocks and quantified to determine the concentration of viral DNA in the original stock. We used the reported molecular weight of Ad5 DNA of about 2.4 x 107 (van der Eb et al., 1969) to calculate the concentration of genomes present in the stock, which was equated to the concentration of virion particles. Titers were then expressed as particles/ml.
Immunofluorescence analysis and microscopy: HeLa cells were grown on cover slips in 35 mm dishes. Cells were either uninfected or infected with wild type Ad5 or the indicated viral mutants. In some cases, the cells were treated with 40 µg/ml α-amanitin (Sigma-Aldrich), a RNA polymerase II inhibitor, 30 min after infection to study the role of transcription in Mdc1 foci formation. Cells were fixed at various hours post infection (hpi) in paraformaldehyde (PFA). In brief, cells were washed in phosphate-buffered saline (PBS; 137mM NaCl, 2.7mM KCl, 8mM
Na2HPO4, 1.46mM KH2PO4), gently fixed with 1% PFA in PBS for 1 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and then fixed with 4% PFA for 10 min. The fixed cells were blocked for 30 min in blocking reagent buffer (100mM Tris [pH 7.5], 150mM NaCl, 0.5%
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Table 1. Status of E4-11kDa and E4-34kDa gene in the E4 mutants used in this study.
Presence (+) or absence (-) of:
______
Virus E4-11kDa/ORF3 E4-34kDa/ORF6
Ad5 + +
H5dl1007 - -
H5dl1010 + -
H5dl1014 - -
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blocking reagent [Amersham]) and incubated with primary antibodies in blocking reagent buffer. The primary antibodies and the dilutions used for immunostaining were rabbit polyclonal anti- Mdc1 (Bethyl Labs) at 1:200, mouse monoclonal antibody against E2-72kDa (kindly provided by A. Levine) at 1:100, and goat polyclonal anti-Mre11 (Santa Cruz Biotechnology) at 1:400. After three 5-min washes in PBS, cells were incubated with appropriate secondary antibodies (Invitrogen) including Alexafluor 594 donkey anti-rabbit IgG (H+L), Alexafluor 488 donkey anti-mouse IgG (H+L), and/or Alexafluor 594 donkey anti-goat IgG (H+L) at dilutions of 1:2000. After three 5-min washes in PBS, the cells on cover slips were mounted on glass slides with Vectashield mounting media (Vector Laboratories, Inc.). The cells were visualized and scored using the 100X objective of a Nikon Eclipse E-400 microscope. Images were captured using SPOT Advanced charge-coupled device and capture software (Diagnostic Instruments Inc.). Representative images were chosen and composite images were assembled using Adobe Photoshop CS5 software.
Western blotting: HeLa cells were grown in 35-mm dishes. Cells were either uninfected or infected with wild type Ad5 and mutant viruses. In some cases, cells were treated with UV (100 mJ/cm2) as indicated. Cells from each 35 mm dish were harvested, washed with ice-cold PBS, and pelleted by centrifugation. Cell pellets were lysed in 200 µl of radioimmunoprecipitation assay lysis buffer (50mM Tris [pH 8.0], 150mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1.0% Igepal CA-630, 0.5% deoxycholic acid) supplemented with protease inhibitors, aprotinin and leupeptin at 5 µg/ml (Amresco), and a phosphatase inhibitor cocktail in accordance with the manufacturer’s instructions (Sigma-Aldrich). Cell lysates were sonicated and total protein levels in samples were measured by Bradford assay using Coomassie Plus protein reagent (Pierce), according to the manufacturer’s instructions. Samples with equal amounts of total protein (50-120 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 5%, 8%, 10% or 15% polyacrylamide gels. The separated proteins were transferred overnight to enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham). The membranes were probed with specific primary antibodies diluted either in 5% nonfat dry milk or 5% bovine serum albumin (BSA) dissolved in 1X Tris-Buffered Saline (0.02M Tris-HCl [pH7.4], 0.136M NaCl, 0.1% Tween-20) (TBST). The primary antibodies used in immunoblotting were mouse monoclonal antibody against E2-72kDa (kindly supplied by A.
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Levine) at 1:1000 dilution, goat polyclonal anti-Mre11 antibody (Santa Cruz Biotechnology) at 1:200, rabbit polyclonal anti-E4-11kDa antibody (a gift from G. Ketner) at 1:700, rabbit monoclonal antibody against Chk1 phosphoserine 345 at 1:1000, mouse monoclonal antibody against Chk1 at 1:1000 (Cell Signaling), rabbit polyclonal antibody against Nbs1 phosphoserine 343 (Santa Cruz Biotechnology) at 1:200, and goat polyclonal against Nbs1 (Santa Cruz Biotechnology) at 1:200. After three 15-min washes in 1X TBST, the protein blots were incubated with horseradish peroxidase-conjugated anti-goat (Santa Cruz Biotechnology), anti- rabbit and anti-mouse (Amersham) secondary antibodies diluted 1:1500 in 5% nonfat dry milk or 5% BSA. After four 15-min washes in 1X TBST, membranes were incubated with ECL reagent (Amersham) to generate chemiluminescence signals, which were subsequently captured on ECL hyperfilm (Amersham). For quantitation of protein levels, the membranes were probed with alkaline phosphatase conjugated anti-goat, anti-rabbit and anti-mouse (Santa Cruz Biotechnology) secondary antibodies at 1:1500 dilution. Protein blots were subsequently incubated with enhanced chemifluorescence substrate (Amersham) and images were captured using a STORM 860 phosphorimager (Molecular Dynamics). The proteins were quantified from captured images using ImageQuant 5.2 (Molecular Dynamics) software.
Viral DNA analysis: Total and nuclear DNA was isolated from uninfected and infected cells as described previously (Weinberg and Ketner, 1986). In brief, cells were washed twice with ice-cold PBS and subsequently harvested and centrifuged to recover cell pellets. For total DNA, cell pellets were lysed in 0.05M Tris [pH 7.8], 0.0025M EDTA, 0.25% SDS containing proteinase K at 0.4 mg/ml. DNA was extracted twice with phenol and chloroform, precipitated with ethanol, and the recovered DNA was dissolved in nuclease-free water. For isolation of nuclear DNA, cell pellets were processed through two rounds of gentle resuspension in lysis buffer (0.14M NaCl, 1.5mM MgCl2, 100mM Tris [pH 8.6], 0.5% Igepal CA-630, 1mM dithiothreitol) to solubilize cytoplasmic membranes, and centrifugation to recover the nuclei. The pelleted nuclei were used to prepare total DNA as described above.
For dot blot analysis, total and nuclear DNA samples were treated with RNase (DNase free) for 1 h at 0.5 µg/ml. Samples were extracted with phenol and chloroform and DNA was precipitated using ethanol. RNase-free DNA samples (2 to10 µg) were denatured by boiling at 100°C and subsequently chilled on ice. DNA samples were adjusted to a final concentration of 24
6X SSC using 20X SSC (3M NaCl, 300mM sodium citrate). The dot blot manifold was set up with 6X SSC presoaked Hybond-N nylon membrane (Amersham) for DNA transfer. DNA samples in 6X SSC were spot transferred to a Hybond-N nylon membrane through the wells of a dot blot manifold under gentle vacuum. DNA was fixed to the membrane by UV cross-linking using a UV transilluminator. The membranes were probed with a radioactive Ad-specific probe as described below for Southern blot analysis.
For Southern blot analysis, 10 µg of total DNA from each sample was digested with EcoRI and subjected to electrophoresis in a 1% agarose gel for 20 h at 20 V. DNA was transferred to Hybond-N nylon membrane (Amersham), which was subsequently hybridized with Ad-specific probe. Ad-specific [32P] probe was synthesized using Ad5 genomic DNA as a template for the multiprime DNA labeling system (GE Healthcare/Amersham) in accordance with the manufacturer's instructions. Hybridization with 5 x 106 cpm/ml probe was performed at 65°C for 20 h. The membranes were subjected to phosphorimaging analysis, and DNA levels were quantified using ImageQuant 5.2 (Molecular Dynamics) software.
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Results
Mdc1 focus formation is correlated with MOI and requires viral genome transcription but not replication. Relocalization of Mdc1 into foci is one of the earliest events observed in activation of the DDR in response to ionizing radiation (reviewed in Coster and Goldberg, 2010). We have previously found that Mdc1 foci formation can be observed by 4 hpi before the onset of viral DNA replication in both Ad5 and E4 mutant infections (Mathew and Bridge, 2007). AdRSVgal lacks E1 and is therefore deficient for both viral early gene expression and DNA replication (Berk et al., 1979). We investigated the distribution of Mdc1 in cells infected with replication defective AdRSVgal in comparison to replication competent wild-type Ad5, and E4 mutant H5dl1007, which replicates its DNA similarly to Ad5 at MOI 30 FFU/cell, to determine whether Mdc1 focus formation is efficient in replication defective infections. Immunofluorescence micrographs showing Mdc1 redistribution into foci in cells infected with the indicated viruses are shown in Fig. 4A. Efficient Mdc1 focus formation was seen in all of the infections. We infected cells at MOIs of 30 and 300 FFU/cell to determine whether there is a correlation between Mdc1 focus formation and levels of input virus. Uninfected cells showed a background level of Mdc1 focus formation of ca. 7%. Mdc1 focus formation was observed in 15 to 20% of the cells infected at MOI 30 FFU/cell with all three viruses tested; this was an increase of at least 2-fold over background (Fig. 4B, top graph). Mdc1 focus formation was observed in 35 to 50% of the cells infected at MOI 300 FFU/cell (Fig. 4B bottom graph). This 6 to 7-fold increase in Mdc1 focus formation over background was observed with each of the viruses tested. These results indicate that the efficiency of Mdc1 foci formation was correlated with levels of virus used to infect the cells and did not depend on viral DNA replication. These results are consistent with the idea that delivery of viral genomes to the cell by infection is sufficient to induce this aspect of the DDR response even in the absence of viral DNA replication.
To confirm that AdRSVgal infections were indeed defective for early gene expression and DNA replication, HeLa cells were infected at a MOI of 30 FFU/cell, and early gene expression was assayed by western blotting using an antibody that detects the viral 72-kDa DNA binding protein produced from E2 (E2-72kDa) and the E4-11kDa protein produced from E4 ORF 3. Figure 5A shows that E2-72kDa is efficiently produced in Ad5 and H5dl1007 infections
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Figure 4. Mdc1 focus formation in Ad-infected cells is correlated with MOI. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1-), at an MOI of 30 or 300 FFU/cell as indicated. The cells were then fixed with paraformaldehyde at 5 hpi and immunostained with antibody against Mdc1. (A) Immunofluorescence micrographs showing Mdc1 redistribution into large foci in cells infected with the indicated viruses at an MOI of 30 FFU/cell. (B) Cell cultures were infected with the indicated viruses, blindly scored for Mdc1 foci, and the percentage of cells with large Mdc1 foci was plotted for infections performed at an MOI of 30 or 300 FFU/cell. Error bars show the standard error of the mean from three independent experiments.
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but is dramatically reduced in AdRSVgal infections, confirming that AdRSVgal is deficient for expression of this protein. E4-11kDa protein levels were reduced in AdRSVgal infections compared to Ad5, but not as dramatically as the E2-72kDa protein (Fig. 5B). These results indicate that AdRSVgal has leaky expression of E4-11kDa even in the absence of functional E1. We next assessed the ability of AdRSVgal to replicate its DNA to determine if the reductions in early gene expression were sufficient to prevent viral DNA replication under our infection conditions. HeLa cells were infected with AdRSVgal at an MOI of 30 or 300 FFU/cell, and total DNA levels at 4 and 24 hpi were measured by dot blot analysis (Fig. 5C). We saw no increase in viral DNA levels between 4 and 24 hpi at either MOI, confirming that AdRSVgal is profoundly replication defective. We also measured levels of total and nuclear DNA in Ad5, H5dl1007, and AdRSVgal infections at 4 hpi to confirm that the mutant viruses can efficiently deliver their DNA genomes to the nucleus. The levels of total and nuclear DNA were similar for all the infections, and AdRSVgal was not deficient for delivery of viral DNA to the nucleus when compared with Ad5 (Fig. 5D).
AdRSVgal has been engineered to produce -galactosidase driven by a Rous sarcoma virus promoter, which is expected to be constitutively active. Since recent results from Karen and Hearing (2011) suggest that transcription-mediated remodeling of the viral chromatin is needed to activate phosphorylated ATM focus formation, we next sought to determine whether active transcription was required for Mdc1 focus formation in infected cells. HeLa cells were infected with Ad5, AdRSVgal or H5dl1007, and immediately treated with α-amanitin to inhibit transcription from the incoming genomes. Inhibiting transcription significantly decreased Mdc1 foci formation in all infections (Fig. 6A and B). In control experiments we found that α-amanitin treatment from 30 min to 10 hpi was sufficient to block E2-72kDa production in Ad5 infections throughout the length of the early phase (Fig. 6C), indicating that the treatment effectively blocked early gene expression. These results suggest that transcription from the viral genome is important for the cell’s ability to respond to infection by forming Mdc1 foci.
AdRSVgal infection fails to activate phosphorylation of Nbs1 and Chk1 and redistributes Mre11 to nuclear tracks. Since AdRSVgal infection induces Mdc1 focus formation (Fig. 4),
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Figure 5. AdRSVβgal is defective for viral early gene expression and viral DNA replication. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1-) at a MOI of 30 FFU/cell. (A and B) Western blotting was performed on extracts prepared at 24 hpi with antibody against the E2-72kDa (72K) (A) and E4-11kDa (11K) (B). (C) Total DNA samples from cells infected with AdRSVβgal at MOI 30 or 300 FFU/cell were prepared at 4 and 24 hpi and analyzed by dot blotting. DNA levels were expressed as the fold difference from AdRSVβgal DNA levels measured at 4 hpi for an MOI of 30 FFU/cell, which was set at 1 (D). HeLa cells were infected with the indicated viruses at an MOI 30 FFU/cell. Total (T) and nuclear (N) DNA samples were prepared at 4 hpi (prior to the onset of DNA replication) and analyzed by dot blotting. DNA levels were expressed relative to the Ad5 levels for both total and nuclear DNA, which were each set at 1. Error bars show the standard errors of the mean from three independent experiments.
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which is an early DDR, we next investigated its ability to induce phosphorylation of Nbs1 and Chk1. In E4 mutant infections Nbs1 and Chk1 represent examples of several substrates that are phosphorylated by the ATM and/or ATR kinases following sensing of DNA damage by the MRN complex (Carson et al., 2003). HeLa cells were either uninfected, or infected with Ad5 or the indicated mutants at a MOI of 30 FFU/cell and then cultured for 22-24 hpi. Protein extracts prepared from these cultures were subjected to SDS-PAGE and western blot analysis using antibodies against phosphorylated Nbs1, or phosphorylated Chk1 (Fig. 7). Ad5 does not efficiently phosphorylate Nbs1 or Chk1 as expected, since its E4 proteins inactivate Mre11 (Carson et al., 2003). The E4 deletion mutant H5dl1007 induced substantial levels of Nbs1 and Chk1 phosphorylation. In contrast, AdRSVgal did not activate phosphorylation of either protein. The levels of unphosphorylated Nbs1 and Chk1 were not significantly affected by infection with these viruses, with the exception of unphosphorylated Nbs1 in Ad5 infections, which was reduced compared to uninfected controls (Fig. 7) due to degradation of MRN complex proteins by Ad5 E4 proteins as shown previously (Carson et al., 2003). Our results indicate that although AdRSVgal infection is sufficient to stimulate Mdc1 foci formation (Fig. 4), it does not activate a full DDR that includes phosphorylation of Nbs1 and Chk1.
We next wished to determine whether Mre11 is affected by AdRSVgal infections. Ad5 produces E4 proteins that inactivate Mre11 by degradation and redistribution thereby preventing activation of many aspects of the cellular DDR. AdRSVgal lacks E1b and therefore cannot make the E1b-55kDa/E4-34kDa complex that targets Mre11 for proteasome-mediated degradation. However, AdRSVgal does express E4-11kDa, albeit at reduced levels compared to Ad5 (Fig. 5B), and could therefore affect the distribution of Mre11. We measured Mre11 levels and its distribution in AdRSVgal infections. HeLa cells were either uninfected or infected with Ad5, H5dl1007, or AdRSVgal at MOI of 30 FFU/cell. Western blotting with Mre11 antibody was performed using extracts prepared at 24 hpi, and the results are shown in Fig. 8A. Mre11 is reduced in Ad5 infections as expected, but the levels of Mre11 are relatively unaffected by infection with either H5dl1007 or AdRSVgal. We next assessed Mre11 localization by immunofluorescence staining of infected cells with antibodies against Mre11 and the viral E2- 72kDa DNA binding protein (Fig. 8B). Mre11 was substantially degraded in Ad5 infected cells.
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Figure 6. Mdc1 focus formation depends on transcription from the viral genome. (A) HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1-) viruses at a MOI of 30 FFU/cell. At 30 min post infection, experimental cells were left untreated or treated with 40 µg/ml α-amanitin (α-am). The cells were fixed with paraformaldehyde at 5 hpi and immunostained with antibody against Mdc1. (B) The cells in each infected culture were blindly scored for Mdc1 focus formation in three independent experiments, and the results were graphed. Error bars show the standard error of the mean. (C) As a control we measured the effect of α- amanitin treatment on Ad5 early gene expression. Ad5 infected HeLa cells were left untreated or treated with α-amanitin from 30 min post infection. Protein extracts prepared at 10 hpi were subjected to western blot analysis with an antibody that detects E2-72kDa (72K) as marker for early gene expression.
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In H5dl1007 infected cells, Mre11 colocalized with viral DNA replication centers containing E2- 72kDa, as shown previously (Stracker et al., 2002). In AdRSVgal infections Mre11 is detected in nuclear tracks. The appearance of Mre11 in tracks in AdRSVgal infections is delayed relative to Ad5 in time course experiments (data not shown), but cells with Mre11 redistributed to nuclear tracks accumulate steadily and by 20 hpi represent the majority of the infected cells. This result indicates that AdRSVgal infection is able to redistribute Mre11 into nuclear tracks, which could potentially impact its ability to activate Nbs1 and Chk1 phosphorylation.
E4-11kDa-mediated redistribution of Mre11 to nuclear tracks is not sufficient to prevent Nbs1 phosphorylation. E4-11kDa-mediated distribution of Mre11 to nuclear tracks affects the ability of ATR to phosphorylate Chk1 but does not prevent the activation of ATM in infections with viruses that express E4-11kDa but lack the ability to degrade the MRN complex (Carson et al., 2009). Nbs1 is potentially a substrate of either the ATM or ATR kinase. We performed infections with E4 mutant H5dl1010 to address the role of Mre11 redistribution in Nbs1 phosphorylation. H5dl1010 lacks an intact gene for E4-34kDa and is unable to degrade Mre11 (data not shown), but expresses E4-11kDa and therefore efficiently redistributes Mre11 to nuclear tracks (Fig. 8B). Western blotting indicated that H5dl1010 did not activate Chk1 phosphorylation, as expected (Carson et al., 2009). However, Nbs1 was phosphorylated in H5dl1010 infections (Fig. 8C) despite efficient Mre11 redistribution (Fig. 8B). This result is consistent with the idea that E4-11kDa-mediated Mre11 redistribution does not prevent ATM activation (Carson et al., 2009) and suggests that ATM-mediated activation of Nbs1 occurs in H5dl1010 infections. Although H5dl1010 induction of Nbs1 phosphorylation was not as strong as in H5dl1007 infections, this is likely because ATR cannot contribute to Nbs1 phosphorylation in H5dl1010 infections. A further implication of this result is that the failure of AdRSVgal to activate Nbs1 phosphorylation is not simply due to E4-11kDa-mediated Mre11 redistribution, since AdRSVgal redistributes Mre11 similarly to H5dl1010 (Fig. 8B) but does not activate Nbs1 phosphorylation (Fig. 8C).
DNA damage triggers can still induce Nbs1 phosphorylation in AdRSVgal infected cells. The failure of AdRSVgal to activate Nbs1 phosphorylation led us to hypothesize that this replication defective virus might be missing an activation trigger that is provided in H5dl1010
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Figure 7. Replication defective AdRSVβgal fails to activate phosphorylation of Nbs1 and Chk1. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1-) at a MOI of 30 FFU/cell for 22 to 24 h. Protein extracts prepared from these cultures were subjected to SDS-PAGE and western blot analyses using antibodies against phosphorylated Nbs1 (pNbs1) (A) or phosphorylated Chk1 (pChk1) (B). Extracts from the same infections were assayed in Western blots using antibodies to detect unphosphorylated epitopes of Nbs1 (A) and Chk1 (B), and actin (middle and lower panels, respectively).
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infections. An alternative possibility is that AdRSVgal infection inactivates the DDRs needed for Nbs1 phosphorylation. To address these possibilities, we exposed AdRSVgal infected cells to UV light to see if these cells were still capable of responding to a known DNA damage trigger. UV treatment induces both ATM (Yajima et al., 2009) and ATR (Cimprich and Cortez, 2008) responses, and cells that lack functional ATM are defective for repair of UV-induced DNA damage (Hannan et al., 2002). HeLa cells were uninfected or infected with Ad5 or AdRSVgal at a MOI of 30 FFU/cell and, at 22 hpi, were either exposed to UV (100 mJ/cm2) or left untreated. We harvested the cultures at 24 hpi, and protein lysates were analyzed by Western blotting using antibody against phosphorylated Nbs1 (Fig. 9). Phosphorylated Nbs1 levels increased in AdRSVgal infected cells treated with UV compared to untreated infections. Ad5 infected cells with or without UV treatment didn’t show any appreciable difference in phosphorylated Nbs1 levels. These results indicate that while Ad5 is capable of preventing UV- induced Nbs1 phosphorylation, presumably through its inactivation of Mre11 by degradation, AdRSVgal is not. Our data suggests that AdRSVgal infected cells are capable of phosphorylating Nbs1 in response to a UV DNA damage trigger and support the idea that AdRSVgal infection by itself fails to provide an activating trigger needed to induce efficient Nbs1 phosphorylation.
Viral DNA replication is important for activating Nbs1 phosphorylation. AdRSVgal is replication defective and fails to induce phosphorylation of Nbs1 or Chk1, while H5dl1010, which replicates its DNA normally at high MOI (Bridge and Ketner, 1989), activated Nbs1 phosphorylation (Fig. 8). We next explored the possibility that viral DNA replication may provide a trigger that is important for inducing these responses. E4 mutant H5dl1007 replicates its DNA similarly to wild-type Ad5 at high MOI (Bridge and Ketner, 1989), but at lower MOIs E4 mutants that lack the genes for both E4-34kDa and E4-11kDa have substantial DNA replication defects (Halbert et al., 1985; Weinberg and Ketner, 1986). We investigated the induction of Nbs1 phosphorylation following low multiplicity infections by H5dl1007 to determine whether viral DNA replication was correlated with inducing Nbs1 phosphorylation. HeLa cells were infected with wild-type Ad5 at an MOI of 30 FFU/cell or H5dl1007 at MOIs of
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Figure 8. Redistribution of Mre11 to nuclear tracks is not sufficient to prevent Nbs1 phosphorylation. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, H5dl1010 or AdRSVβgal (E1-) viruses at a MOI of 30 FFU/cell. (A) Western blotting was performed on extracts prepared at 24 hpi from the indicated infections, using antibody against Mre11 (top panel). Phosphorimaging analysis of three independent blots was performed and the results plotted (bottom panel). Error bars show the standard error of the mean. (B) Mre11 localization was determined by double immunofluorescence staining of cells infected with the indicated viruses and fixed at 20 to 24 hpi and immunostained with Mre11 and E2-72kDa antibodies. (C) Western blotting was performed on extracts prepared at 24 hpi from the indicated infections, using antibody against phosphorylated Nbs1 (pNbs1) (top panel) or phosphorylated Chk1 (pChk1) (bottom panel).
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30, 3, 1, and 0.3 FFU/cell. Even at the lowest MOI tested with H5dl1007, > 70% of the cells were infected, as determined by immunofluorescence detection of E2-72kDa at 24 hpi (data not shown). At the low MOIs of 1 and 0.3 FFU/cell, E2-72kDa staining was primarily diffuse and replication foci containing this protein were rarely observed (data not shown). Total DNA was isolated from infected HeLa cells at 22 to 24 hpi for Southern blot analysis (Fig. 10A, top panel). The levels of viral DNA were quantified by phosphorimaging analysis (Fig. 10A, bottom graph). The H5dl1007 DNA levels were similar to Ad5 at a MOI of 30 FFU/cell and 30-fold lower at a MOI of 3 FFU/cell and were not detectable above the background signal of uninfected HeLa cells at a MOI of 1 or 0.3 FFU/cell. We next measured the levels of phosphorylated Nbs1 to assess activation of cellular DDR proteins in H5dl1007 infections performed at different MOIs. Cell extracts were prepared at 22 hpi for western blot analysis using antibody against phosphorylated Nbs1. The results are shown in Fig. 10B. H5dl1007 infection done at an MOI of 30 or 3 FFU/cell resulted in a substantial increase in the levels of phosphorylated Nbs1 compared to the uninfected HeLa cell control. In contrast, H5dl1007 infections carried out at a MOI of 0.3 FFU/cell did not activate Nbs1 phosphorylation.
We next addressed the possibility that a certain threshold of viral DNA ends might be needed to activate a DDR and that, when lower levels of virus are used for infection, replication is required to produce this threshold number of ends. We performed dot blot analysis to measure the levels of viral DNA present in cells infected with replication defective AdRSVgal at an MOI of 30 FFU/cell or with H5dl1007 at an MOI of 3.0 FFU/cell to address this issue. DNA samples were prepared at 4 and 22 hpi to measure viral DNA levels before and after the typical onset of viral replication, which occurs at about 12 hpi (Fig. 11). We observed a modest increase in H5dl1007 DNA levels of 3 and 4-fold between 4 and 22 hpi at an MOI of 3.0 FFU/cell (Fig. 10C), and H5dl1007 induced phosphorylation of Nbs1 at this MOI (Fig. 10B). In contrast, input AdRSVgal DNA levels at 4 hpi at a MOI of 30 FFU/cell were already higher than the level achieved by H5dl1007 replication for 22h in cells infected at 3.0 FFU/cell. AdRSVgal DNA levels were actually less at 22 hpi than at 4 hpi, confirming that no DNA replication occurred during AdRSVgal infections at this MOI (Fig. 5C). AdRSVgal did not induce phosphorylation of Nbs1 at an MOI 30 FFU/cell (Fig. 10B). The observation that H5dl1007 induces a damage response at low MOI despite achieving fewer DNA templates following replication than were 41
Figure 9. UV treatment of AdRSVβgal infected cells activates phosphorylation of Nbs1. HeLa cells were uninfected (UI) or infected with Ad5, or AdRSVβgal (E1-) at an MOI of 30 FFU/cell, and after 22h cultures were either exposed to UV (100 mJ/cm2) as indicated or left untreated. Cultures were harvested at 24 hpi, and protein lysates prepared and analyzed by western blotting using antibody against phosphorylated Nbs1 (pNbs1). A representative western blot is shown in panel A. Phosphorimaging analysis of three independent blots was performed and a graph of the results is shown in panel B. Error bars show the standard errors of the mean.
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present following infection with AdRSVgal at high MOI argues that achieving a threshold level of viral DNA is not sufficient to activate Nbs1 phosphorylation.
AdRSVgal lacks E1, raising the possibility that its failure to activate some DDRs could be due to its failure to express E1 genes. We addressed this by studying E4 mutant H5dl1014, which has a more substantial DNA replication defect than H5dl1007 (Bridge et al., 1993) but still carries an intact E1 region. H5dl1014 is derived from wild type Ad5 and carries two deletions that destroy all the E4 ORFs except ORF 4 (Bridge and Ketner, 1989). HeLa cells were infected with H5dl1007 or H5dl1014 at an MOI of 200 or 800 viral particles/cell. Total DNA was prepared at 4 and 24 hpi and analyzed by dot blotting (Fig. 10D). At an MOI 200 particles/cell (top panel) H5dl1014 and H5dl1007 had similar levels of input DNA at 4 hpi, which is before the onset of DNA replication (the ratio of H5dl1014/H5dl1007 DNA levels was ~ 1.1). This indicates that H5dl1014 and H5dl1007 infections delivered equal amounts of viral DNA to the cell. H5dl1007 DNA levels increased 27-fold, while H5dl1014 DNA levels increased only 2.7-fold, by 24 hpi. In parallel infections, total cell extracts were prepared at 24 hpi for Western blot analysis with an antibody against phosphorylated Nbs1. Extract prepared from UV-treated cells was used as a control for Nbs1 activation. At 200 particles/cell, H5dl1014 did not activate Nbs1 phosphorylation, whereas H5dl1007 showed a substantial increase in phosphorylated Nbs1 levels compared to the uninfected control (Fig. 10E, top panel). Since these viruses have identical E1 regions, the major difference between the two infections is deficient DNA replication by H5dl1014. When similar experiments were performed using 800 particles/cell, we saw significant increases in viral DNA levels between 4 and 24 hpi (Fig. 10D, bottom panel) and activation of Nbs1 phosphorylation (Fig. 10E, bottom panel) in both H5dl1014 and H5dl1007 infections. H5dl1014 and H5dl1007 showed a 22- and 165-fold increase in DNA levels, respectively, at this MOI. Taken together, these results suggest that replication of viral DNA provides a trigger that induces phosphorylation of the DDR protein Nbs1.
Our results indicate that phosphorylation of Nbs1 occurs only in E4 mutant infections capable of replicating viral DNA. Therefore, we next wanted to investigate whether the onset of H5dl1007 DNA replication correlates with the activation of host DDR proteins in time course
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Figure 10. Viral DNA replication is important for activating Nbs1 phosphorylation. HeLa cells were either uninfected (UI) or infected with wild-type Ad5, AdRSVgal (E1-), H5dl1007, or H5dl1014 at the indicated MOIs and cultured for 22-24 hpi. The MOIs used in FFU/cell are in parentheses. (A) Southern blot analysis was performed with 10 g of EcoRI-digested DNA prepared from each infection. The EcoRI C fragment used for comparison of viral DNA levels is shown in the top panel. Viral DNA levels were quantified by phosphorimaging analysis (lower graph). E4 mutant H5dl1007 DNA levels are expressed as the fraction of the Ad5 level, which was set at 1. Error bars indicate the standard deviations from three independent experiments. (B) Protein extracts from infected cells were subjected to SDS-PAGE and western blot analysis using antibody against phosphorylated Nbs1. (C) Cells were infected with AdRSVgal (E1-) and H5dl1007 at MOIs of 30 and 3 FFU/cell, respectively. DNA levels from the indicated infections and times (hpi) were measured by dot blot analysis and are expressed as the fraction of AdRSVgal DNA levels measured at 4 hpi, which was set at 1. Error bars indicate the standard deviations from three independent experiments. (D) Total DNA samples were prepared from the indicated infections and hpi, and the samples were analyzed by dot blotting. DNA levels were expressed as the fold difference from H5dl1007 DNA levels measured at 4 hpi after infection with 200 particles/cell (top graph) or 800 particles/cell (bottom graph), which was set at 1. Error bars show the standard errors of the mean from three independent experiments. (E) Protein extracts from the indicated infections were subjected to SDS-PAGE and western blot analysis using antibody against phosphorylated Nbs1 (pNbs1). In panels D and E MOIs were expressed as virion particles/cell (see Materials and Methods). UV treated uninfected cells were used as a positive control for induction of pNbs1.
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experiments. HeLa cells were infected with H5dl1007 at 30 FFU/cell. Total DNA was isolated at 0, 4, 8, 16 and 22 hpi for Southern blot analysis. Figure 11A shows the findings from a phosphorimaging analysis of E4 mutant viral DNA levels expressed as the fold increase over the background levels seen in uninfected controls. We find that H5dl1007 DNA levels increase by 10-fold between 12 and 16 hpi indicating the onset of viral DNA replication. In a parallel experiment, total cell extracts were prepared from H5dl1007 infected cells at the times indicated for western blot analysis with antibodies against phosphorylated Nbs1 and Chk1. The levels of phosphorylated Nbs1 and Chk1 were similar to background levels seen in uninfected cells (0 hpi) until ~ 12 hpi, and then increased by 8 to 10-fold between 12 and 16 hpi (Fig. 11B and C). This correlates very well with the onset of viral DNA replication (Fig. 11A), indicating that viral DNA replication coincides with phosphorylation of the DDR proteins Nbs1 and Chk1.
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Figure 11. E4 mutant-induced phosphorylation of Nbs1 and Chk1 correlates with the onset of viral DNA replication. HeLa cells were infected with H5dl1007 at 30 FFU/cell. Total DNA was isolated at 0, 4, 8, 16 and 22 hpi for Southern blot analysis and DNA levels were quantified by phosphorimaging analysis. (A) E4 mutant DNA levels were measured as fold increase over the background level detected in samples prepared from uninfected cells, which was set at 1. In a parallel experiment, protein lysates were prepared from H5dl1007- infected cells at the times indicated and processed for western blot analyses using antibodies against phosphorylated Nbs1 (B) and Chk1 (C). Protein levels were quantified by phosphorimaging analysis. The levels of phosphorylated Nbs1 (pNbs1) and Chk1 (pChk1) are expressed as fold increase compared to the level detected in uninfected HeLa cells, which was set at 1.
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Discussion
The induction of cellular DDRs has been extensively investigated. DSBs produced by ionizing radiation are sensed by the MRN complex, which activates kinases critical for both cell cycle checkpoint activation and for repair of the broken DNA (Harper and Elledge, 2007; Lee and Paull, 2005). Ad infection also induces cellular DDRs (Carson et al., 2003). Ad infection and replication presents the cell with both unusual protein-linked DNA ends and ssDNA replication intermediates that could be interpreted by the cell as signs of DNA damage. We have used a replication-defective Ad vector, AdRSVβgal, to study the activation of DNA repair responses during infections that efficiently deliver Ad genomes to the nucleus but are then incapable of carrying out viral DNA replication (Fig. 5). To our surprise, although AdRSVβgal infection induced Mdc1 foci formation (Fig. 4), it was deficient for activation of other aspects of the DDR, including phosphorylation of Nbs1 and Chk1 (Fig. 7).
Mdc1 is one of the earliest proteins to be recruited to the site of DNA breaks induced by ionizing radiation. We have previously found that the MRN sensor complex is needed for efficient Mdc1 focus formation in Ad-infected cells (Mathew and Bridge, 2007) and that both Mre11 and Mdc1 are bound to viral DNA at early times after infection in chromatin immunoprecipitation (ChIP) experiments (Mathew and Bridge, 2008). Mdc1 focus formation is correlated with MOI (Fig. 4), indicating that it could be a response to the increase in incoming genomes. Ad genomes are packaged in the virion with the core proteins pV and pVII (Corden et al., 1976; Mirza and Weber, 1982; Vayda et al., 1983). The viral genome bound to core proteins and covalently linked terminal proteins is then delivered to the nucleus during infection (Haruki et al., 2003; Spector et al., 2003). ChIP experiments indicate that the Ad genome interacts with histones and that the genomic template for early transcription contains a mix of cellular histones and the viral core protein pVII. During the process of early transcription the genome undergoes chromatin remodeling and pVII is replaced by acetylated histones (Komatsu et al., 2011). Karen and Hearing (2011) have studied the formation of foci containing phosphorylated ATM (pATM) in E4 mutant infections, and find that pATM colocalizes with Ad genomes that no longer contain the viral core DNA binding protein pVII. Furthermore, pATM foci are reduced in cells treated with a transcription inhibitor and after infection by Ad E1 mutants that fail to activate any of the viral genome’s transcription units. These observations indicated that early transcription-mediated 50
uncoating of pVII from the viral genome is important for pATM focus formation. Likewise, we find that transcription inhibition can also interfere with Mdc1 focus formation in Ad5, E4 mutant, and AdRSVβgal infections (Fig. 6). Although we cannot rule out the possibility that early gene expression per se is required for activating Mdc1 focus formation, the observation that AdRSVβgal induces efficient Mdc1 focus formation (Fig. 4) despite the absence of E1 genes and poor activation of other early genes (Fig. 5), argues against this. Rather, we consider it more likely that transcription-mediated chromatin remodeling is important for Mdc1 focus formation, similar to the pATM foci identified by Karen and Hearing (2011). It is possible that the ability of the MRN complex to sense and respond to incoming viral DNA genomes depends on their chromatin configuration and that exchange of the viral core proteins for a more cell-like chromatin configuration involving cellular histones is critical for the MRN complex to recognize the viral genome as “DNA damage” and activate responses such as Mdc1 and pATM focus formation.
Although AdRSVβgal was able to activate Mdc1 foci formation, which is an early DDR event during Ad infection, we found that it was unable to trigger phosphorylation of either Nbs1 or Chk1 (Fig. 7). Typically, these proteins are substrates of the ATM and/or ATR kinases and are phosphorylated following ionizing radiation. Recently Carson et al. (2009) have shown that redistribution of the MRN complex to nuclear tracks by the E4-11kDa protein can prevent ATR- mediated signaling, including phosphorylation of Chk1. AdRSVβgal can produce E4-11kDa (Fig. 5), and redistribute Mre11 to nuclear tracks (Fig. 8), so it is therefore possible that this explains its failure to activate Chk1. This possibility is supported by the observation that H5dl1010 infections also fail to phosphorylate Chk1 (Fig. 8C). H5dl1010 carries a deletion affecting E4-34kDa, and like AdRSVβgal, does not degrade Mre11 (data not shown) but efficiently redistributes it to nuclear tracks (Fig. 8B). However, H5dl1010 induced phosphorylation of Nbs1, whereas AdRSVβgal did not (Fig. 8C). This raises the possibility that either AdRSVβgal fails to phosphorylate Nbs1 because it has inactivated DDR responses in infected cells, or alternatively, the AdRSVβgal life cycle may be so defective that it fails to produce triggers necessary to activate aspects of the DDR needed for efficient Nbs1 phosphorylation. Interestingly, UV treatment of AdRSVβgal infected cells induced Nbs1 phosphorylation, although the levels were not quite as high as in UV treated uninfected cells
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(Fig. 9). This could be because ATR contributes to Nbs1 phosphorylation in UV-treated uninfected cells, whereas ATR responses are likely to be inactivated by Mre11 redistribution in AdRSVβgal infections (Figs. 7, 8) (Carson et al., 2009). Nevertheless, this result indicates that AdRSVβgal infection does not block the ability of cells to respond to UV-induced DNA damage and suggests that AdRSVβgal infections fail to provide a necessary trigger for activating Nbs1 phosphorylation. We think it is likely that replication-defective infections may also fail to provide triggers needed for activating Chk1 phosphorylation, but this must be confirmed with a vector that lacks E1 as well as the gene encoding the E4-11kDa protein (E4 ORF3), and is therefore not capable of inactivating ATR by Mre11 redistribution to nuclear tracks.
We consistently find that Ad mutant infections that do not result in significant viral DNA replication fail to induce phosphorylation of Nbs1. This includes AdRSVβgal infections, H5dl1007 infections performed at a low MOI, and infections with H5dl1014, which is more defective for DNA replication than H5dl1007 (Fig. 10). We do not find evidence that a threshold level of incoming DNA templates is required to activate Nbs1 phosphorylation (Fig. 10C). Since AdRSVβgal lacks the E1 region in addition to being defective for DNA replication, it is possible that the E1 proteins themselves are important for activating some DDRs (Cuconati et al., 2003). However, H5dl1014 and H5dl1007 both carry an intact E1 region, yet H5dl1014 still failed to activate phosphorylation of Nbs1 under infection conditions that resulted in similar delivery of viral genomes to the cell but very little H5dl1014 DNA replication compared with H5dl1007. When cells were infected with four times more H5dl1014 and H5dl1007, we observed significant DNA replication as well as efficient Nbs1 phosphorylation in infections with both viruses (Fig. 10D and E). We also find that phosphorylation of both Nbs1 and Chk1 is coincident with the onset of viral DNA replication in E4 mutant-infected cells in time course experiments (Fig. 11). Our results are most consistent with a model in which Nbs1 and possibly also Chk1 phosphorylation requires replication of input viral DNA. Karen et al., (2009), concluded that input genomes of a replication defective E4 mutant were sufficient to induce ATM phosphorylation. However, we note that their replication defective E4 mutant, dl355/inORF3, was actually able to increase DNA levels >100-fold between 8 and 24 hpi in their DNA replication assay. Although this is certainly defective compared to wild-type infections, it is comparable to the 165-fold increase in DNA levels we observed between 4 and 24 hpi with a
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similar E4 mutant, H5dl1007, which was sufficient to induce significant Nbs1 phosphorylation (Fig. 10). We suggest that relatively modest levels of viral DNA replication are sufficient for activating DNA replication dependent DDRs, and while these levels are achieved in typical infections with defective E4 mutants, they are not achieved in AdRSVβgal infections, which show no increase in viral DNA levels between 4 and 24 hpi (Figs. 5 and 10).
Why would viral DNA replication be important for activating cellular DDRs? The ATR kinase is known to respond to ssDNA (Zou and Elledge, 2003), so it is possible that the ssDNA intermediates produced during Ad replication are an important part of the mechanism involved in triggering ATR activation. Nichols et al. (2009) have shown that widespread phosphorylation of the histone H2AX during Ad infection requires viral DNA replication, and their results were consistent with a role for the ATR kinase in this response. ATM is thought to respond to the presence of DSBs (Harper and Elledge, 2007; Lee and Paull, 2005; Lee and Paull, 2007), and if incoming linear viral DNA genomes mimic such breaks, it is possible that only ATM responses would be active in replication defective AdRSVβgal infections. However, we have data suggesting that Mdc1 foci still form in cells that lack ATM (D. Gautam and E. Bridge, unpublished data), indicating that this response may be independent of ATM in Ad- infected cells. Preliminary data suggests that while we see significant phosphorylation of ATM in both H5dl1007 and H5dl1010 infections, we see relatively little ATM phosphorylation in AdRSVβgal infections (A. Prakash and E. Bridge, unpublished data). Although Nbs1 is not exclusively a substrate of the ATM kinase, it is noteworthy that we also failed to see Nbs1 phosphorylation in AdRSVβgal infections (Fig. 7). Thus, it is possible that delivery of viral genomes to the nucleus in the absence of DNA replication is not sufficient for full activation of either ATM or ATR. In vitro studies have shown that ATM activation requires linear dsDNA and the MRN complex (Lee and Paull, 2005). Interestingly, these authors also found that the ability of the MRN complex to unwind DNA ends, thereby producing ssDNA, was critical for ATM activation. It is possible that the presence of 5’ terminal proteins on Ad genomes interferes with efficient DNA unwinding by the MRN complex, thereby making the virus genome a poor activator of ATM even though it is linear dsDNA. If this is the case, Ad DNA replication may be necessary to produce the ssDNA triggers needed for both ATM and ATR activation. Our results are consistent with the idea that incoming genomes may induce a limited set of DDR responses, including
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Mdc1 focus formation and the induction of focal concentrations of γH2AX (Nichols et al., 2009), while widespread production of γH2AX (Nichols et al., 2009) and induction of Nbs1 and possibly also Chk1 phosphorylation may require viral DNA replication. In conclusion, it will be informative to dissect out the ability of cellular DDR proteins to react to replication-defective and replication-competent Ad infections since this may provide new insights into how DNA damage is sensed and transduced to activate cellular responses.
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Acknowledgements
We are very grateful to Gary Ketner and Arnold Levine for providing antibodies used in this study. We thank Michel Perricaudet for providing AdRSVβgal. We also thank Gary Janssen for critically reading the manuscript and all the members of our laboratory for their suggestions and support.
This research was supported by the National Cancer Institute (grant CA82111) and awards from Miami University.
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Adenovirus DNA replication amplifies ATM dependent signaling and contributes to the induction G2/M cell cycle arrest during activation of the DNA damage response
Anand Prakasha, Stephanie Swedika, and Eileen Bridgeab#
aDepartment of Microbiology
32 Pearson Hall
Miami University
Oxford OH 45056 bCell, Molecular and Structural Biology Program
#Corresponding author
Tel: 513-529-7264
Fax: 513-529-2431
Email: [email protected]
Manuscript in preparation
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Abstract Adenovirus (Ad) mutants that lack early region 4 (E4) activate a cellular DNA damage response (DDR) that includes activation and foci formation of multiple DDR proteins. E4 proteins inactivate the Mre11, Rad50, Nbs1 (MRN) sensor complex, which is responsible for sensing DNA damage and transducing damage signals to downstream effector kinases involved in DNA repair and cell cycle checkpoint arrest. Replication competent E4 mutant infection activates DDRs that involve phosphorylation of the effector kinase Ataxia-Telangiectasia Mutated (ATM) and its substrates, and can induce G2/M cell cycle arrest. We investigated the ability of replication defective and replication competent Ad mutants to activate ATM dependent signaling and G2/M cell cycle arrest, to further understand the role of DNA replication in these responses. Ad infection induced early focal accumulation of DDR proteins such as Mre11, a component of the MRN sensor complex, phosphorylated ATM (pATM), phosphorylated Chk2 (pChk2), and 53BPI, independent of the replication status of mutants studied. However, Mre11 and pATM foci were transient in replication defective infections and were only maintained in infections with replication competent mutants. Viral DNA replication was correlated with amplification of pATM levels as well as its substrate, pChk2. Furthermore, we found that G2/M cell cycle arrest was not activated by a replication defective mutant or a mutant expressing the E4orf3 encoded 11kDa protein. Our results suggest that the initial induction of DDR foci does not require viral DNA replication. In contrast, viral DNA replication is important for maintenance of DDR proteins at viral replication centers, amplification of pATM and pChk2, and G2/M cell cycle arrest.
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Introduction
Ad is a linear double stranded DNA virus that inactivates cellular DDRs to allow successful viral replication and progeny virus production. Ad E4 gene products inactivate Mre11, which is an important component of the MRN DNA damage sensor complex (Stracker et al., 2002). The E4orf3 encoded 11kDa (E4-11kDa) protein inactivates Mre11 by redistributing it away from viral replication centers (Evans and Hearing, 2005). The E4orf6 encoded 34kDa gene product, (E4-34kDa), makes a complex with E1b-55kDa to inactivate Mre11 via proteasome- mediated degradation (Stracker et al., 2002). By interfering with the MRN complex, E4 proteins prevent activation of proteins involved in DNA repair, cell cycle checkpoint arrest, and apoptosis (Weitzman and Ornelles, 2005). Mutants that fail to express these proteins therefore activate a cellular DDR in response to virus infection (Stracker et al., 2002; Carson et al., 2003; Carson et al., 2009).
The cellular DDR can be activated by a variety of exogenous (e.g. ionizing radiation (IR), ultraviolet radiation (UV), radiomimetic drugs, and virus infection) as well as endogenous (e.g. metabolic reactive oxygen free radicals, meiotic recombination, and replication fork collapse) triggers (De Bont and van Larebeke, 2004; Lilley et al., 2010). IR induces double strand breaks (DSBs) in DNA which are sensed by the MRN complex. The MRN complex is important for recruiting the kinase ATM, which phosphorylates chromatin binding proteins such as H2AX, creating a platform for recruiting multiple DDR proteins to the DSBs. These proteins accumulate at the site of the DSB and can be microscopically visualized as discrete nuclear foci (Lou et al., 2006; Bekker-Jensen et al., 2005; Stucki et al., 2005). A variety of protein-protein interactions regulate focal assembly, disassembly, and residence time of DDR proteins at DSBs (reviewed in Polo and Jackson, 2011). Although the function of DDR foci is not well understood, it is speculated that they promote DNA repair and cell cycle checkpoint signaling (Coster and Goldberg, 2010).
ATM activates a number of downstream effector proteins involved in cell cycle arrest (Shiloh, 2013). Cellular progression to mitosis is regulated by the concerted action of Cdc2 and Cyclin B1 (Graves et al., 2001; Abraham, 2001) and is halted during the DDR. ATM and the related phosphoinositide 3-kinase (PI-3 kinase), ATM and Rad3 related (ATR), phosphorylate
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Chk2 and Chk1 (pChk1), respectively, to induce cell cycle arrest. The pChk1 and pChk2 kinases inactivate the phosphatase Cdc25c (Matsuoka et al., 1998). This blocks dephosphorylation of Cdc2, which is required for progression to mitosis. During mitosis, histone 3 (H3) is phosphorylated at serine 10 and the presence of phosphorylated H3 (pH3) is considered a marker for the onset of mitosis (Hans and Dimitrov, 2001). ATM dependent G2/M arrest correlates with decreased pH3 (Xu et al., 2001).
The role of Ad viral DNA replication in ATM dependent signaling and G2/M cell cycle arrest is not well understood. E1 mutants are replication defective and fail to efficiently express viral early genes (Berk, 1979). E4 deletion mutant H5dl1007 (1007) lacks the ability to make the E4-34kDa and E4-11kDa proteins, and induces cellular DDRs (Stracker et al., 2002; Carson et al., 2003) and G2/M cell cycle arrest (Cherubini et al., 2006). E4 mutant H5dl1010 (1010) lacks the 34kDa gene but expresses E4-11kDa, and a similar mutant is known to activate ATM but not ATR responses (Carson et al., 2003; 2009). Both 1007 and 1010 replicate viral DNA efficiently following high multiplicity of infection (MOI) (Bridge and Ketner, 1989). In the absence of viral DNA replication, Ad mutants induce focal accumulation of phosphorylated H2AX (γ-H2AX) (Nicholas et al., 2009), Mdc1 (Prakash et al., 2012), and pATM (Karen and Hearing, 2009). The pATM foci co-localize with incoming viral DNA genomes (Karen and Hearing, 2011), suggesting that the foci assemble on incoming viral genomes. Viral DNA replication begins around 8-10 hours after high MOI Ad infection. In time course experiments performed with wild type Ad5, pATM foci disappear as viral replication centers develop (Gautam and Bridge, 2013). The loss of pATM foci in Ad5 infections is the result of E4 protein-mediated inactivation of the cellular DDR. In contrast, we have shown that pATM foci continue to co-localize with developing viral DNA replication centers in E4 mutant infections (Gautam and Bridge, 2013). These findings suggest a possible role for viral DNA replication in maintenance of pATM foci in E4 mutant infections. Nichols et al. (2009) found that a replication defective mutant induces focal accumulation of γ-H2AX, but viral DNA replication was important for widespread phosphorylation and amplification of H2AX. Furthermore, we have shown that a replication competent E4 mutant induces phosphorylation of Nbs1 while a replication defective mutant does not (Prakash et al., 2012). These reports suggest that viral DNA replication maybe important for amplifying levels of phosphorylated Nbs1 and H2AX, both of which are ATM substrates. ATM
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and its effector substrate Chk2 are phosphorylated in E4 mutant infection (Carson et al., 2003), but the contribution of viral DNA replication to maintenance of DDR foci and amplification of activated pATM and pChk2 has not been studied.
Here we investigated DDR foci formation, phosphorylation of ATM and its substrate Chk2, and induction of G2/M cell cycle arrest in replication competent and replication defective Ad infections to clarify the role of viral DNA replication in these DDRs. Our results indicate that the focal accumulation of Mre11, Mdc1, pATM, pChk2, and 53BPI occurs independently of viral DNA replication. However, viral DNA replication is important for continued maintenance of these foci. We observe that phosphorylation of ATM and Chk2 is amplified upon viral DNA replication. Furthermore, we found that initial pATM and pChk2 foci formation in E1- infections and widespread nuclear accumulation of activated ATM and Chk2 in 1010 infections is not sufficient for activating G2/M cell cycle arrest.
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Materials and Methods
Cells and viruses. HeLa, E4 mutant complementing W162 cells (Weinberg and Ketner, 1983), and E1 mutant complementing HEK293 cells were grown in monolayer cultures in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 U/ml penicillin and 10 µg/ml streptomycin. Wild type Ad5 and E4 mutants were propagated on W162 cells. E4 mutants used in the present study include H5dl1007 (referred to here as 1007) and H5dl1010 (referred to here as 1010) (Bridge and Ketner, 1989). AdRSVβgal, which substitutes the β-galactosidase gene in place of viral E1 region under the control of the Rous sarcoma virus promoter (Stratford-Perricaudet et al., 1992) (referred to here as E1-), was grown on HEK293 cells. Ad5 titers were determined on both HEK293 and W162 cells and expressed as fluorescent focus forming units (FFU)/ml (Phillipson, 1961). E1- and E4 mutant titers were determined on HEK293 and W162 cells, respectively. Ad5 titers on both HEK293 and W162 cells were used to calculate a conversation factor which was used to determine the W162 titer equivalent for AdRSVβgal. HeLa cells were infected at different MOIs using W162 titers of Ad5 and the mutants.
Immunofluorescence analysis and microscopy: HeLa cells were grown on coverslips in 35 mm dishes and were either uninfected or infected with wild type Ad5 or the indicated mutants. At various hours post infection (hpi), immunofluorescence staining and microscopy was performed as previously described (Gautam and Bridge, 2013). In some of the immunostaining experiments, 5% FBS in PBS was used as the blocking reagent.
The following primary antibodies were used at specific dilutions in immunostaining: rabbit polyclonal anti-Mdc1 (Bethyl Labs) at 1:200, goat polyclonal anti-Mre11 (Santa Cruz Biotechnology) at 1:100, rabbit polyclonal anti-72K (a gift from T. Linné) at 1:1500, mouse monoclonal anti-ATM phospho epitope S1981 (anti-pATM, Novus) at 1:200, rabbit polyclonal anti-53BP1 (Abcam) at 1:100, rabbit polyclonal anti-Chk2 phospho epitope T68 (anti-pChk2, Abcam) at 1:40; and mouse monoclonal anti-histone H3 phospho epitope S10 (anti-pH3, Millipore) at 1:50. The following secondary antibodies (Invitrogen) were used: Alexafluor 594 donkey anti-rabbit IgG (H+L), Alexafluor 488 donkey anti-mouse IgG (H+L), and/or Alexafluor 594 donkey anti-goat IgG (H+L) at dilution of 1:250.
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Images were captured with a confocal laser scanning microscope (Olympus FV500), using a 100X oil immersion objective, and Fluoview software. The images from Alexa 488 and Alexa 594 channels were sequentially scanned to check bleed through between the channels. Representative images were chosen and composite images were assembled using Adobe Photoshop CS5 software.
Western blotting: HeLa cells grown in 35-mm dishes were either uninfected or infected with wild type Ad5 or Ad mutants. At the desired time, cells were washed and harvested with ice-cold PBS. The cell pellets were lysed in 200 µl of radioimmunoprecipitation assay lysis buffer (50mM Tris [pH 8.0], 150mM NaCl, 0.1% sodium dodecyl sµlfate [SDS], 1.0% Igepal CA-630, 0.5% deoxycholic acid) supplemented with protease inhibitors aprotinin and leupeptin at a final concentration of 5 µg/ml (Amresco), and phosphatase inhibitor (PhosStop) as per the manufacturer’s instructions (Roche). After 15 min incubation on ice, cell extracts were sonicated at 25% amplitude for 5 sec on/off for three times. The extracts were then centrifuged and supernatants were collected. The protein concentration of the supernatants was measured by Bradford assay using Coomassie Plus protein reagent (Pierce), as per the manufacturer’s instructions.
For pH3 and H3 western blots, HeLa cells grown on 60 mm dish were either uninfected or infected with different mutants. At 12 hpi, HeLa cells were washed and harvested with ice- cold PBS. The cell suspension was subjected to centrifugation to collect the cell pellet. For nuclei extraction, the cell pellet was resuspended in 250 µl of Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X-100 (v/v), protease inhibitors aprotinin and leupeptin at a final concentration of 5 µg/ml (Amresco), and phosphatase inhibitor (PhosStop) as per the manufacturer’s instructions (Roche). The extract was incubated on ice for 15 min followed by centrifugation at 2000 rpm for 15 min to pellet the nuclei. The supernatant was removed from the samples and discarded. The nuclear pellet was washed in 125 µl of TEB and centrifuged as before. The nuclear pellet was resuspended in 70 µl of 0.2 N HCl and incubated overnight at 4°C for histone extraction. The samples were centrifuged at 2000 rpm for 15 min. The supernatant was removed and collected to determine protein content using the Bradford assay.
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Samples containing equal amounts of total protein were separated by SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Different percentages of polyacrylamide were used depending upon the size of the protein to be detected. The separated proteins were transferred overnight to enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham). The membranes were blocked in 5% nonfat dry milk in 1X TBST for 2-3 hrs. Then, the membranes were probed with specific primary antibodies diluted either in 5% nonfat dry milk or 5% bovine serum albumin (BSA) in 1X TBST for overnight incubation at 4 °C. The following primary antibodies were used at specific dilutions: rabbit polyclonal anti-E4-11kDa (a gift from G. Ketner) at 1:700, rabbit polyclonal anti-Chk2 phospho epitope T68 (anti-pChk2, Abcam) at 1:1000, mouse monoclonal anti-Chk2 (Santa Cruz Biotechnology) at 1:200, rabbit monoclonal anti-ATM phospho epitope S1981(anti-pATM, Abcam) at 1:2000, rabbit monoclonal anti-ATM (Abcam) at 1:5000, mouse monoclonal anti-β-actin (Santa Cruz Biotechnology) at 1:200, mouse monoclonal anti-histone H3 phospho epitope S10 (anti-pH3, Millipore) at 1:1000, and mouse monoclonal anti-histone H3 (Santa Cruz Biotechnology) at 1:200. The membrane was washed with 1X TBST three times for 15 min and was incubated with horseradish peroxidase-conjugated anti-goat, anti-rabbit, or anti-mouse (Santa Cruz Biotechnology) secondary antibody diluted 1:1500 in 5% nonfat dry milk in 1X TBST. After three 15-min washes in 1X TBST, the membrane was incubated with ECL Plus reagent (Thermoscientific) to generate chemiluminescence signals, which were subsequently captured on ECL hyperfilm (Amersham).
Viral DNA analysis: Total DNA was extracted from uninfected and Ad infected cells as described previously (Weinberg and Ketner, 1986). In brief, cells were harvested in ice cold PBS and the cells were then pelleted by centrifugation. The cell pellet was incubated at 37°C for 1h in cell lysis buffer with proteinase K (0.05M Tris [pH 7.8], 0.0025M EDTA, 0.25% SDS containing proteinase K at 0.4 mg/ml). The lysate was subjected to two rounds of phenol and chloroform extraction. The collected supernatant was ethanol precipitated, centrifuged, and the pelleted nucleic acid was dissolved in nuclease-free water. Samples were treated with RNase (DNase free) for 1 h at 0.5 µg/ml concentration, followed by two rounds of phenol and chloroform extraction, and ethanol precipitation of the DNA. The recovered DNA was then analyzed by dot blotting. RNase-free DNA samples were denatured by heating to 100°C for 10 min, transfered to
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ice and adjusted to a final concentration of 6X SSC using 20X SSC (3M NaCl, 300mM sodium citrate). DNA samples were transferred to 6X SSC presoaked Hybond-N nylon membrane (Amersham) using a dot blot manifold under gentle vacuum. The DNA was cross-linked to the membrane using a UV transilluminator and the membrane was subjected to hybridization with Ad-specific [32P] probe that was synthesized from Ad5 genomic DNA using the multiprime DNA labeling system (GE Healthcare/Amersham) as per the manufacturer's instructions. The membrane was incubated with 5 x 106 cpm/ml of Ad-specific [32P] probe overnight at 65°C. The amount of radioactivity bound to the membrane was determined by phosphorimaging using a STORM 860 phosphorimager (Molecular Dynamics) and DNA levels were quantified using ImageQuant 5.2 (Molecular Dynamics) software.
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Results
Focal accumulation of pATM is not maintained in the absence of DNA replication or in the presence of E4 11kDa
The focal accumulation of γ-H2AX (Nicholas et al., 2009), Mdc1 (Prakash et al., 2012), and pATM (Karen and Hearing, 2009) occurs in response to unreplicated Ad genomes. However, pATM foci co-localize with developing viral DNA replication centers in E4 mutant infections (Gautam and Bridge, 2013), suggesting a possible role for viral DNA replication in the maintenance of pATM foci. E4-11kDa induces Mre11 redistribution (Stracker et al., 2002) and the MRN complex is required to localize pATM to viral replication centers (Gautam and Bridge, 2013). We compared focal accumulation of pATM in replication defective E1- and replication competent E4 mutants, in time course experiments to investigate the role of viral replication and E4-11kDa expression in maintenance of pATM foci. E1- is defective for both viral early gene expression and DNA replication (Berk et al., 1979). The 1007 mutant lacks the entire E4 region including the genes for both E4-34kDa and E4-11kDa and therefore induces cellular DDRs (Stracker et al., 2002; Carson et al., 2003). E4 mutant 1010 lacks the gene for E4-34kDa but expresses E4-11kDa and redistributes Mre11 (Stracker et al., 2002). HeLa cells were either uninfected or infected with E1-, 1007, 1010, or wild type Ad5, at a MOI of 30 FFU/cell. Infected cells were fixed at 5 and 12 hpi, which is before or after viral DNA replication, respectively. The fixed cells were subjected to immunofluorescence staining using antibodies to pATM, Mre11, Mdc1, and the viral E2-72kDa DNA-binding protein (72kDa). Images were captured using confocal microscopy. At 5 hpi, pATM foci were observed in all the infections, and co-localized with other DDR proteins including Mre11 and Mdc1 (Fig. 12A). We did not observe viral DNA replication centers marked with the 72kDa antibody at 5 hpi (data not shown), as expected. Uninfected cells showed a background level of pATM foci in approximately 4% of cells. Infection with E1-, 1007, 1010, and Ad5 induced a 4-5 fold increase in pATM foci formation to 19%, 23%, 22%, and 22% of the infected cells, respectively. The presence of pATM foci prior to the onset of viral DNA replication and in replication defective E1- indicates that focal accumulation of pATM is independent of viral DNA replication. We studied pATM localization
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Figure 12. Focal accumulation of pATM is not maintained in the absence of DNA replication or in the presence of E4-11kDa. HeLa cells were either uninfected (UI) or infected with Ad5, 1007, 1010, or E1- at an MOI of 30 FFU/cell for the times indicated and then fixed and immunostained with antibodies against pATM, Mdc1, Mre11, or the viral DNA binding protein 72kDa to detect viral DNA replication centers. (A) Immunofluorescence confocal micrographs showing pATM (green), Mdc1 (red), and Mre11 (green) of uninfected cells or cells infected with the indicated virus at 5 hpi. Infected cells were blindly scored for cells with pATM foci and the percentage of cells in the culture with pATM foci is shown for each virus infection under the respective panels. (B) Immunofluorescence confocal micrographs showing pATM (green), E2-72kDa (red), and Mre11 (red) immunostaining of infected cells at 12 hpi. (C) HeLa cells were infected with Ad5 and the indicated mutants at an MOI of 30 FFU/cell. At 12 hpi, cells were harvested and processed for western blot analysis using antibody against E4-11kDa. Levels of the control protein β-actin are also shown.
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after the onset of viral DNA replication at 12 hpi to determine the contribution of viral DNA replication to the maintenance of pATM foci. E4 mutant 1007 maintained focal accumulation of pATM at developing DNA replication centers detected with anti-72kDa antibodies (Fig. 12B). The pATM foci co-localized with Mre11, as expected (Stracker et al., 2002; Carson et al., 2003; Fig. 12B, panels j and n). In contrast, replication defective E1- did not maintain pATM foci (Fig. 12B, panels a and i). Replication centers containing 72kDa were not observed in E1- (panel e), as expected, and dot blot analysis confirmed that E1- did not show any increase in viral DNA levels from 4 to 12 hpi (data not shown). Wild type Ad5 did not maintain pATM foci due to degradation of the MRN sensor complex by E4 proteins (Stracker et al., 2002; Carson et al., 2003), as reported previously (Fig. 12B, panels d, l, and p). E4 mutant 1010 makes the E4-11kDa protein which is responsible for Mre11 redistribution. Mre11 was redistributed to track like structures in 1010 infection, as expected. Although 1010 did not maintain pATM in foci, pATM was present and showed widespread nuclear staining that did not colocalize with viral DNA replication centers (Fig. 12B, panels c and g).
Since E1- carries the gene for E4-11kDa and we have previously shown leaky expression of E4-11kDa in E1- infections at 24 hpi (Prakash et al., 2012), we assessed the level of E4- 11kDa by western blot at 12 hpi to determine if E4-11kDa could be responsible for loss of pATM foci in E1- infection. As expected, E4-11kDa was not observed in 1007 but was abundantly expressed in 1010 and Ad5 infections (Fig. 12C). No expression of E4-11kDa was observed in E1- infection, which was consistent with failure of this virus to redistribute Mre11 in immunofluorescence experiments (Fig. 12B, panel m). This shows that the loss of pATM foci in E1- infection is not due to E4-11kDa mediated Mre11 redistribution, and more likely reflects the failure of E1- to replicate viral DNA. These findings indicate that pATM foci formed prior to DNA replication in response to the delivery of viral genomes are not maintained in the presence of E4-11kDa or in the absence of viral DNA replication.
Viral DNA replication correlates with amplification of pATM and its downstream substrate pChk2
Since E1- induces focal accumulation of pATM in the absence of viral DNA replication (Fig. 12A), we asked whether ATM dependent downstream signaling is detected in E1-
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Figure 13. ATM substrates are detected in foci in the absence of viral DNA replication but an increase in phosphoprotein levels is not detected in western blot analysis. HeLa cells were either uninfected (UI) or infected with E1- at an MOI of 30 FFU/cell and then fixed at 5 hpi. The cells were immunostained with antibodies against pATM and pChk2 (left panel) or 53BPI (right panel). (B) HeLa cells were either uninfected (UI) or infected with E1- at an MOI of 30 or 150 MOI FFU/cell as indicated, and extracts were prepared at 5 hpi. Western blotting was performed using antibody against pATM, ATM, and, β-actin (left panel) or pChk2, Chk2, and, β-actin (right panel). An extract prepared at 12 hpi with 1010 at a MOI of 30 FFU/cell was used as a positive control, since it activates pATM.
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infections. In IR, ATM phosphorylates the downstream kinase Chk2 at T68 (Ahn et al., 2000), and both pATM and pChk2 are present in foci (Wilson and Stern, 2008). ATM also induces focal accumulation of 53BPI (DiTullio et al., 2002). We analyzed focal accumulation of pChk2 and 53BPI relative to pATM in HeLa cells that were either uninfected or infected with E1- at a MOI of 30 FFU/cell for 5 h. We observed that pChk2 and 53BPI co-localize with pATM in foci in E1- infected cells (Fig. 13A). Since only 19-22 % of infected cells showed pATM foci (Fig. 12A), we asked whether this level of activation could be detected as an increase in protein levels measured by western blotting. HeLa cells were infected with E1- at an MOI of 30 or 150 FFU/cell and cell lysates prepared at 5hpi were analyzed by western blot using pATM and pChk2 antibodies. Interestingly, the levels of pATM and pChk2 were not significantly different from the uninfected control, even at the higher MOI of 150 (Fig. 13B), which is expected to show a higher percentage of cells in the culture with DDR foci (Prakash et al, 2012). Infection with E1- had little effect on the levels of unphosphorylated ATM and Chk2 (Fig. 13B). Taken together these findings suggest that although E1- induces significant focal accumulation of pATM and pChk2 detected by immunofluorescence staining, a significant increase in cellular levels of phosphorylated ATM and Chk2 is not detected by western blot in the absence of DNA replication.
E4 mutant 1010 is unable to maintain pATM in foci but instead shows widespread nuclear pATM localization following viral DNA replication (Fig. 12B). This raises the possibility that viral DNA replication is important for amplifying ATM dependent signaling. We next asked whether phosphorylation of ATM and its substrate Chk2 are increased during viral DNA replication. HeLa cells were either uninfected or infected with replication defective E1- and replication competent 1010 at a MOI of 30 FFU/cell and immunofluorescence staining was performed at 12 hpi. In E1- infected cells, we did not observe foci with pATM or pChk2 (Fig. 14A) indicating that the failure of E1- to maintain pATM foci (Fig. 12B) also results in failure to maintain pChk2 foci. In contrast, 1010 induced abundant widespread nuclear staining of both pATM and pChk2 (Fig. 14A). Western blot analysis of protein samples prepared at 12 hpi confirmed that 1010 infection induced robust phosphorylation of ATM and Chk2 while replication defective E1- showed little if any induction of these proteins (Fig. 14B). These results show that pATM and pChk2 levels are significantly amplified in replication competent 1010
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Figure 14. Viral DNA replication amplifies ATM dependent signaling. HeLa cells were either uninfected (UI) or infected with 1010 or E1- at an MOI of 30 FFU/cell. (A) At 12 hpi, cells were fixed and immunostained with antibodies against pATM and pChk2. (B) Protein extracts prepared from these cultures at 12 hpi were subjected to SDS-PAGE and western blot analysis using antibodies against pATM, ATM, and β-actin (left panel) or pChk2, Chk2, and, β- actin (right panel). (C) HeLa cells were either uninfected (UI) or infected with 1007 at an MOI of 30 FFU/cell and subsequently incubated with the ATM kinase inhibitor KU60019 from 2–12 hpi. Cells not treated with the inhibitor were incubated with an equivalent amount of the DMSO solvent used to dissolve the inhibitor, as a control. Western blotting was performed on extracts prepared at 12 hpi with antibody against pChk2, Chk2, and, β-actin.
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infections. To assess viral DNA levels, we performed dot blot analysis at 4 and 12 hpi to measure input and replicated viral DNA respectively. We observed that viral DNA levels did not change between 4 and 12 hpi in E1- infection, indicating no viral DNA replication had occurred. In contrast, 1010 DNA levels increased ~100 fold between 4 and 12 hpi (data not shown). We also found that the ATM inhibitor, KU60019 (Golding et al., 2009), prevented induction of pChk2 in E4 mutant infections, confirming that pATM is important for this activity (Fig. 14C). Our findings support the idea that although replication defective E1- can activate focal accumulation of pATM and pChk2 at early times (Fig. 13A), viral DNA replication is needed to significantly amplify pATM and pChk2 levels at later times in infected cultures.
G2/M cell cycle arrest occurs in 1007, but not in E1- and 1010 infections.
E4 mutant infection activates ATM and ATR dependent signaling (Carson et al., 2003; Carson et al., 2009) and induces G2/M checkpoint arrest (Cherubini et al., 2006). In response to IR, ATM dependent G2/M arrest correlates with decreased histone H3 phosphorylation (Xu et al., 2001). We investigated G2/M cell cycle arrest using an antibody against the mitotic marker pH3. HeLa cells were either uninfected or infected with E1-, 1007, or 1010 at a MOI of 30 FFU/cell. Infected cells were harvested at 12 hpi and western blotting was performed with anti- pH3. The levels of pH3 were substantially reduced in 1007 compared to uninfected cells, indicating fewer mitotic cells in 1007 infections. In contrast, E1- and 1010 infections showed no decrease in pH3 levels, which were similar to uninfected cells (Fig. 15A). In parallel experiments, we determined the number of mitotic cells by scoring pH3 positive cells in immunofluorescence experiments. We observed a significant decrease in the percent of mitotic cells in 1007 (1.6%), but not in 1010 (4%), or E1- (5.4%) infected cultures compared to the uninfected control (5%) (Fig. 15B). We observed that E1- and 1010 induce initial foci formation of pATM and pChk2 (Fig. 13A). 1010 also induces widespread nuclear localization of pATM and pChk2 (Fig. 14A). Taken together, these findings indicate initial DDR foci formation in E1- and widespread nuclear accumulation of phosphorylated ATM and Chk2 in 1010 is not sufficient for G2/M cell cycle arrest.
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Figure 15. G2/M cell cycle arrest occurs in 1007 but not in 1010 or E1- infections. HeLa cells were either uninfected (UI) or infected with 1007, 1010, or E1- at an MOI of 30 FFU/cell. (A) Western blot analysis was performed on histone extracts (see Materials and Methods) prepared at 12 hpi from the indicated infections using antibody against histone H3 phospho epitope Ser10 (pH3) and histone H3 (H3). (B) At 12 hpi, cells were fixed and immunostained with antibodies against pH3. The cells were blindly scored for pH3 staining in three independent experiments, and results are graphed. Error bars show the mean and standard deviations.
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Discussion
In this study, we report that the initial accumulation of multiple DDR proteins in foci following Ad infection is independent of viral DNA replication. However, viral DNA replication is required for amplification of pATM and pChk2 levels, and for the maintenance of DDR proteins in foci that colocalize with developing viral DNA replication centers. In agreement with previous findings (Carson et al., 2009), we find that the presence of E4-11kDa does not inhibit ATM or Chk2 phosphorylation, but prevents focal accumulation of these proteins at DNA replication centers. Finally, we show that E1-, which does not replicate its DNA, and 1010, which does not maintain DDR proteins at viral replication centers due to expression of E4- 11kDa, also fail to induce G2/M cell cycle arrest.
In Ad infection, input viral genomes co-localize with pATM foci (Karen and Hearing, 2011). Here we find that pATM co-localizes in foci with multiple other DDR factors including Mre11, Mdc1, pChk2, and 53BPI (Figs. 12A and 13A) as well as γH2AX and RNF8 (data not shown) before the onset of viral DNA replication. These observations confirm that foci induced by Ad infection are similar in composition to foci induced by IR. It has been previously shown that transcription-mediated chromatin remodeling of the input viral genome is required for pATM and Mdc1 foci formation (Karen and Hearing, 2011; Prakash et al., 2012). All Ad viruses we tested induced DDR foci at 5 hpi before the onset of viral DNA replication, and replication defective E1- induced initial DDR foci formation as efficiently as replication competent 1007 (Fig. 12A). These observations show that initial DDR foci formation is independent of viral DNA replication. A replication defective HSV-1 mutant also induces focal accumulation of multiple DDR proteins that co-localize to the incoming viral genome (Lilley et al., 2011), providing evidence that unreplicated genomes trigger similar responses in infections with other viruses. However, foci formation in the case of HSV-1 is independent of transcription mediated genome remodeling (Lilley et al., 2011). Taken together, these observations indicate that DDR foci are induced in the absence of viral DNA replication, and DDR proteins accumulate at incoming transcriptionally remodeled Ad genomes.
Although Ad infection induces DDR foci formation independently of viral DNA replication, levels of activated pATM and its downstream substrates remain low in the absence
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of viral DNA replication (Figs. 13 and 14; Nichols et al., 2009; Prakash et al., 2012). In IR, the DDR is activated in dose-dependent manner (van Veelen et al., 2005), and while low doses of IR induce γH2AX foci formation with barely detectable levels of γH2AX observed by western blot analysis, high doses of IR activate robust levels of γH2AX detectable by the appearance of foci and by western blot (Taneja et al., 2004). These results suggest that increasing the level of DNA damage also increases the level of activated DDR proteins. In Ad infection, DNA replication results in an increase in the number of linear ds viral DNA genomes, which could mimic DSBs. This increase in the level of “DNA damage” events could be responsible for amplifying the intensity of the DDR induced by viral infection. However, it is also possible that the process of viral DNA replication could provide separate triggers to amplify DDRs independently of an increase in viral genome copy number. We and others have shown that increasing viral genome copy numbers by increasing MOI induces an increase in percentage of cells with DDR foci, but this is not sufficient to detect increases in γH2AX (Nichols et al., 2009) and pNbs1(Prakash et al., 2012) levels by western blotting. In contrast, robust phosphorylation of γH2AX and Nbs1 correlates with viral DNA replication (Prakash et al., 2012; Nichols et al., 2009), suggesting that the process of viral DNA replication rather than a simple increase in genome copy number is important for DDR amplification. Here, we have observed that replication defective E1- induces pATM and pChk2 foci (Fig. 13A) but in the absence of viral DNA replication, an increase in the levels of these proteins is not detected by western blot analysis (Fig. 13B). This was true even when the MOI was increased from 30 to 150 FFU/cell (Fig. 13). In contrast, viral DNA replication by 1010 induces a robust increase in the levels of pATM and pChk2, which are readily detectable by western blot analysis (Fig. 14B). Based on previous findings (Prakash et al., 2012; Nichols et al., 2009), we think it is likely that the process of viral DNA replication supplies additional triggers needed to amplify the DDR. Interestingly, HPV-infected, undifferentiated keratinocytes, which do not replicate viral DNA, show focal accumulation of pATM and pChk2, while differentiated keratinocytes capable of HPV DNA replication, amplify pATM, pChk2, and pNbs1 levels (Moody and Laimins, 2009). It is speculated that the HPV E1 helicase involved in viral DNA replication could amplify ATM-dependent signaling in response to viral DNA structures generated as a result of viral DNA replication (Kadja et al., 2007; Sakakibara et al., 2011), providing another example of how the process of viral DNA replication could trigger DDR signaling. 78
Our findings are consistent with the hypothesis that DDR foci formation occurs following transcriptional remodeling of the incoming Ad genome (Fig. 12; Karen and Hearing, 2011), which as linear ds DNA, may mimic DSBs and activate ATM dependent signaling. The DDR could then be amplified by the process of viral DNA replication, which results in synthesis of a large amount of ssDNA and unusual replication intermediates such as pan-handle structures (Challberg and Kelly, 1989). These unusual DNA structures could potentially serve as triggers for further activation of ATM and ATR dependent signaling (Carson et al., 2003; Nichols et al., 2009; Prakash et al., 2012; Fig. 14). The DDR in IR is complex and depends upon factors such as IR dose, chromatin organization, status of transcription and replication, and chromatin modifications (reviewed in Polo and Jackson, 2011). Thus, the use of Ad mutants with limited gene expression and replication capabilities provides a unique opportunity to investigate the role of processes such as genome transcription and replication in DDR activation.
The focal accumulation of DDR proteins at viral replication centers is a common theme in many different virus infections such as SV-40 (Shi et al., 2005), HPV (Moody and Laimins, 2009), and HSV-1 (Wilkinson and Weller, 2004). In HPV infection, pATM and pChk2 foci localize to HPV DNA and these foci persist at viral DNA replication centers (Gillespie et al., 2012), suggesting the possibility that maintenance of DDR factors requires viral DNA replication. Here we observed that the focal accumulation of Mre11, pATM, and pChk2 is maintained at viral replication centers in 1007 infection (Fig. 12B; data not shown). Surprisingly, pATM and pChk2 foci are lost between 5 and 12 hpi by replication defective E1- (Fig. 12). The loss of DDR foci at DSBs in IR occurs when the DSB is repaired (Rothkamm and Lobrich, 2003; Bouquet et al., 2006). Thus it is possible that the loss of pATM and pChk2 foci in E1- by 12 hpi could indicate successful “repair” of viral genomes to form concatemers. This hypothesis is currently being tested. While it is possible that leaky expression of E4-11kDa by E1- could contribute to the disassembly of early DDR foci by inducing Mre11 redistribution, this seems unlikely since E4-11kDa expression was not detected by 12 hpi after focal disassembly had already occurred (Fig. 12). Rather, we think it is likely that the lack of viral DNA replication in E1- infection deprives cells of a continuous supply of activation triggers needed to maintain activated DDR proteins in foci.
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Replication competent E4 deletion mutants maintain Mre11 at viral replication centers (Stracket et al., 2002), activate ATM and ATR dependent signaling (Carson et al., 2003; Carson et al., 2009), and induce G2/M cell cycle arrest (Cherubini et al., 2006). We observed that replication defective E1- and replication competent mutant 1010 do not induce G2/M cell cycle arrest, whereas E4 deletion mutant 1007 does (Fig. 15). E1- induces focal accumulation of DDR proteins but foci are not maintained (Fig. 12) and levels of pATM and pChk2 are not amplified (Fig. 14). Since E1- fails to induce G2/M cell cycle arrest, this suggests that the initial focal accumulation of DDR proteins observed in E1- infections is not sufficient to activate cell cycle arrest. Although 1010 is able to efficiently activate pATM and pChk2 it does not maintain these proteins at viral DNA replication centers due to its expression of E4-11kDa. Interestingly, this mutant also failed to trigger G2/M arrest. This could indicate that activation of ATM is not sufficient for G2/M cell cycle arrest, if ATM is not localized at viral replication centers. Alternatively, since E4-11kDa expression by 1010 is expected to inactivate ATR (Carson et al., 2009), it is possible that E4 mutant induced G2/M cell cycle arrest is ATR- rather than ATM- dependent. We are currently testing these possibilities. Nevertheless, our findings indicate that expression of E4-11kDa by 1010 prevents G2/M arrest, and are consistent with the idea that maintenance of Mre11 at viral replication centers is required for downstream events leading to G2/M cell cycle arrest.
In conclusion, our results support the idea that transcriptionally remodeled incoming genomes induce the initial focal accumulation of activated DDR proteins, and that levels of these proteins are amplified during viral DNA replication. Viral DNA replication dependent amplification of DDRs and the maintenance of Mre11 at viral DNA replication centers are important for G2/M cell cycle arrest. Further studies are needed to determine how viral DNA replication amplifies ATM dependent signaling and the importance of ATM and/or ATR dependent signaling in Ad-induced G2/M cell cycle arrest. Understanding the mechanistic basis for Ad-induced DDRs and G2/M cell cycle arrest will help contribute to our understanding of the molecular mechanisms involved in regulating DDR pathways.
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Concluding remarks
Virus manipulation of cellular DDR
Recent studies show that many DNA and RNA viruses induce cellular DDRs (reviewed in Xiaofei and Kowalik, 2014). DDR induction by viruses is a complex process. Some viruses activate while others inhibit DDR pathways to facilitate viral replication and growth. For instance HPV infection activates ATM and Chk2 dependent signaling which is required for viral DNA replication (Moody and Laimins, 2009). In contrast, wild type Ad5 inhibits ATM and ATR dependent signaling to facilitate efficient viral DNA replication (Carson et al., 2003; Gautam and Bridge, 2013). Also, some viruses selectively inhibit or activate different components of DDR pathways. Herpes simplex virus (HSV-1) disables ATR dependent signaling (Mohini et al., 2010) but activates ATM dependent signaling which is required for viral DNA replication (Lilley et al., 2005) and growth (Li et al., 2008). Thus, viruses have evolved complex mechanisms to manipulate cellular DDR pathways for their own benefit (Chaurushiya and Weitzman, 2009). As replication and growth of many viruses is regulated by the DDR, targeting DDR pathways provides a unique opportunity for treatment of virus induced infectious diseases.
Cellular DDR activation as an anti-oncogenic and anti-viral mechanism
Cells have the intrinsic capacity to sense damaged DNA induced by endogenous and exogenous agents. Under ideal conditions, the cellular DDR pathway induces cell cycle arrest, successfully repairs the damaged DNA, and if irreparable, will induce apoptosis. Thus, the cellular DDR is needed to maintain genomic integrity, which when compromised leads to genomic instability and cancer.
The activation of DDRs serves as a barrier to tumor formation. DDR signaling is observed in precancerous lesions of human breast, urinary bladder, lung, and colon (Bartkova et al., 2005; Gorgoulis et al., 2005). In precancerous lesions, DNA replication stress, either through excessive replication or through induction of DSBs, activates DDR-induced cell cycle arrest and apoptosis. This DDR-based cellular barrier is often inactivated when precancerous lesions progress to cancerous lesions. It is argued that cells with DSBs that do not undergo cell cycle arrest or apoptosis leads to loss of genetic regions as these cells progress through mitosis. Some
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of the lost regions may correspond to genes involved in cancer suppression such as p53. Furthermore, DDR components such as ATM and p53 are frequently mutated in a large number of human cancers (Negrini et al., 2010; Rivlin et al., 2011) and p53 mutant mice are susceptible to carcinogenesis (Attardi, 2004; Ishikawa et al., 2005). Taken together, these findings indicate that cells with DNA damage are released from the suppressive effect of cell cycle arrest and apoptosis in the absence of functional DDRs. These findings support the idea that the DDR represents an early barrier to oncogenesis (Halazonetis et al., 2008) and needs to be inactivated to induce carcinogenesis.
The p53 protein regulates a critical tumor suppressor pathway and upon activation p53 induces transcriptional regulation, cell cycle arrest, and apoptosis of neoplastic or virus infected cells (Haupt et al., 2003). The p53 protein is commonly inactivated in virus infections such as Ad and HPV (O’Shea and Fried, 2005), which respectively cause transformation of host cells and carcinogenesis. In Ad infection, p53 is inactivated either by E4 34kDa - E1b 55kDa- mediated proteasomal degradation (Querido et al., 2001) or by E4-11kDa-induced silencing of p53 dependent promoters (Soria et al., 2010). The HPV E6 protein inactivates p53 by proteasomal degradation (Scheffner et al., 1990). Although inactivation of p53 alone is not sufficient, it is a major factor in HPV carcinogenesis (Feller et al., 2010), suggesting that inactivation of p53 is a viral strategy of DDR inactivation to avoid anti-tumor responses such as apoptosis. The knowledge gained from these studies can be used for therapeutic intervention. The p53 protein is negatively regulated by murine double minute 2 (Mdm2) which suppresses p53-induced cell cycle arrest and apoptosis (Chen et al., 1996). Therefore increasing p53 activity by antagonizing Mdm2 is a novel cancer therapeutic strategy. The Mdm2 antagonist Nutlin exerts its anti-oncogenic effect by at least two different mechanisms: p53-dependent cytotoxic effects on leukemic cells, and p53-independent inhibition of the growth of tumor stromal and vascular cells required for cancer propagation. Thus, the Mdm2 antagonist Nutlin has been proposed as a cancer therapeutic agent for non-viral (Secchiero et al., 2008; Cao et al., 2005) and viral tumors (Sarek et al., 2007; Forte and Luftig, 2009).
The cellular DDR has also emerged as an intrinsic defense against infection with some viruses. The cellular DDR is induced at early times in infection with viruses such as HSV-1 and Ad (Turnell and Grand, 2012; Weitzman et al., 2010). Both viruses must overcome DDR 82
induced blocks to viral DNA replication in order to successfully replicate their genomes and complete their life cycle. To do so, these viruses employ regulatory proteins that interfere with the cellular DDR. E4 proteins from wild type Ad5 inactivate ATM and ATR signaling (Stracker et al., 2002; Carson et al., 2003; Carson et al., 2009) and facilitate efficient viral DNA replication (Gautam and Bridge, 2013). ATM-dependent accumulation of the cellular E3 ubiquitin ligases RNF8 and RNF168 is observed on incoming HSV-1 genomes (Lilley et al., 2010; 2011). RNF8 and RNF168 cause the ubiquitination of H2A (Huen et al., 2007; Doil et al., 2009), which is implicated in transcriptional silencing of damaged DNA (Shangbag et al., 2010). The HSV-1 immediate early protein, ICP0, degrades RNF8, subverts RNF8-dependent silencing, and facilitates viral DNA replication (Lilley et al., 2011). Thus, viral proteins regulate the cellular DDR to facilitate viral replication and completion of the viral life cycle. The lessons learned from these viruses can be used in treatment of cancer cells. Viral proteins that inactivate DDR proteins represent therapeutic alternatives for tumors that are refractory to other therapies. Ad E4orf6 expression radiosensitizes tumor cell lines by interfering with the activity of DNA-PK, a major kinase involved in NHEJ (Hart et al., 2005). Furthermore, Ad E4orf6 induces apoptosis by translocation of apoptosis-inducing factor from mitochondria to the nucleus (Hart et al., 2007). HSV-1 ICP0 protein expression also inhibits DNA-PK and induces apoptosis (Hadjipanayis and Deluca, 2005). The radiosensitization and apoptosis activities of HSV-1 ICP0 have been used for treatment of radioresistant tumors such as human glioblastomas (Hadjipanayis et al., 2008). Therefore, understanding how viral proteins manipulate the cellular DDR allows these proteins to be used as tools for developing cancer therapies against viral and non-viral tumors.
Triggers for DDR activation in virus infection
DDR activation by viruses involves formation of foci similar to IR-induced foci and phosphorylation of multiple DDR proteins. Ad infection induces the focal accumulation of Mdc1 (Mathew and Bridge, 2007), γH2AX (Nichols et al., 2009), and pATM (Karen and Hearing, 2009) before viral DNA replication, and the phosphorylation of several DDR proteins after viral DNA replication (Stracker et al., 2002; Carson et al., 2003). These results suggest the requirement of different triggers for DDR activation. The incoming viral genome, action of viral proteins, and production of replicated viral DNA could all serve as possible triggers for DDR activation (Nikitin and Luftig, 2011; Weitzman et al., 2010). 83
The incoming Ad viral genome represents exogenous DNA that could potentially activate a DDR. We and others have shown that Ad infection induces focal accumulation of DDR factors such as Mre11, pATM, Mdc1, γH2AX, pChk2, and 53BPI in replication defective and competent mutants as well as wild type Ad5 infections, before the onset of viral DNA replication (Chapter I and II; Karen and Hearing, 2011; Nichols et al., 2009). These findings suggest that the unreplicated viral genome is sufficient to induce DDR foci. Although the injection of linearized plasmid to the nucleus activates a DDR (Huang et al., 1996), delivery of the incoming Ad genome is not sufficient for DDR activation. In Ad infection, the incoming viral genome enters coated with viral core binding proteins, including protein VII, and it has been suspected that this organization of viral chromatin is inhibitory for DDR activation. Foci of the pATM protein co- localize with incoming viral genomes (Karen and Hearing, 2011), but pATM and Mdc1 foci are dependent upon genome transcription (Karen and Hearing, 2011; Chapter I and II). Transcription removes protein VII from the viral chromatin and it is replaced with acetylated histones (Komatsu et al., 2011). The linear viral genome bound to histones resembles damaged cellular chromatin and is likely to be a trigger for DDR activation. HSV-1 infection also induces recruitment of DDR proteins such as γH2AX, Mdc1, 53BPI, and BRCA1 at the site of the incoming viral genome (Lilley et al., 2011). The HSV-1 genome enters the nucleus as naked viral DNA and therefore can act as a direct DDR-activating substrate without transcription remodeling, although it is not known whether naked viral DNA or viral DNA in context of cellular histones is important for DDR activation in HSV-1 infection. In summary, unreplicated Ad and HSV genomes are sufficient to activate DDR foci formation.
Viral proteins can activate DDR pathways by multiple mechanisms, both direct and indirect. The HPV E7 protein directly interacts with ATM and activates ATM and Chk2 phosphorylation in undifferentiated keratinocytes (Moody and Laimins, 2009). Viral proteins use various mechanisms to activate the DDR indirectly. First, viral proteins push the cell cycle into S-phase, which results in excess production of replication intermediates and stalled replication forks, all of which could activate the DDR. This is the most common indirect mechanism of DDR activation. The EBNA2 and LMP2 proteins of EBV are implicated in E2F-dependent c- myc transcription upregulation and induction of S phase in infected B cells (Nikitin et al., 2010). Also, Kaposi-Sarcoma Virus’s produces a viral protein that mimics a cellular cyclin and activates
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cyclin dependent kinase-6 to push the cell into S phase (Koopal et al., 2007). In infections with both of these viruses, phosphorylation and foci formation of pATM, pChk2, and 53BPI have been observed (Nikitin et al., 2010; Koopal et al., 2007). Second, SV-40 Large T antigen uses a unique DDR activation mechanism in which it binds to the mitotic spindle checkpoint kinase Bub1. The Bub1 protein blocks anaphase progression because chromatid kinetochores lack bipolar attachment to microtubule spindles and this results in failure of chromatid separation to separate daughter cells (Meraldi and Sorger, 2005). The Large T antigen interaction with Bub1 results in genetic instability manifested as changes in chromosomal numbers such as aneuploidy and tetraploidy (Hein et al., 2009) which contributes to DDR induction. Third, the Ad E1a protein activates viral early transcription, it binds to Rb and induces S-phase progression (Howe and Bayley, 1992) and also induces ATM and H2AX phosphorylation (Cuconati et al., 2003). How E1a activates ATM and H2AX is still unclear. We think it is likely that E1a contributes to Ad induction of DRRs through its ability to induce transcription mediated remodeling of the viral genome as described above (Karen and Hearing, 2011). We have shown that our E1- mutant that has the E1 genetic region substituted with the β-galactosidase gene under the control of Rous Sarcoma virus (Stratford-Perricaudet et al., 1992), induces Mdc1 foci in a transcription dependent manner (Chapter I). As pATM foci co-localize with Mdc1 (Chapter II), this further supports the idea that transcription of the Ad viral genome is sufficient to induce transcription and induction of DDR foci. Thus, our results suggest that E1A has an indirect role in DDR activation via transcription-mediated chromatin remodeling, although an additional role for E1A in DDR induction via pushing cells into S-phase is certainly not ruled out. Overall, viral proteins induce cellular DDR either by direct interaction with DDR proteins, or indirectly by inducing S- phase and replication stress, interaction with a protein involved in mitotic spindle checkpoint, and alteration of cellular chromatin organization. Thus, viral proteins use several mechanisms to induce cellular DDR that are independent of viral DNA replication.
Viral DNA replication can result in the production of ssDNA, dsDNA, and unusual nucleic acid structures, all of which could serve as triggers to activate a cellular DDR. ATM dependent signaling is activated in response to lytic viral DNA replication in EBV infection and newly synthesized viral DNAs are thought to provide activation triggers for these responses (Kudoh et al., 2005). Similarly, viral DNA replication in HPV induces robust ATM-dependent
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phosphorylation of Nbs1, Chk2, and BRCA1 (Moody and Laimins, 2009). The single strand intermediates and episomal structures generated as a result of viral DNA amplification are speculated to activate ATM-dependent signaling (Kadja et al., 2007). A replication competent Ad E4 mutant induces ATM and ATR signaling (Stracker et al., 2002; Carson et al., 2003). We observed that replication competent E4 mutant infection induced phosphorylation of ATM and its substrates Nbs1, Chk2 and the ATR substrate Chk1 but these phosphorylation events were not observed in a replication defective mutant (Chapter I and II), suggesting that viral DNA replication is a likely trigger for these phosphorylation events. It is possible that an increase in viral genome copy number as a result of viral DNA replication could induce DDR events. However, we and others have shown that increasing viral genome copy numbers by increasing MOI is not sufficient to detect increases in γH2AX, pNbs1, pATM and pChk2 levels which are only observed upon viral DNA replication (Nichols et al., 2009; Chapter I; Chapter II). This suggests that the process of viral DNA replication rather than a simple increase in genome copy number is important for DDR amplification. Taken together, these findings suggest that viral DNA replication may provide a variety of replication intermediates that serve as triggers for cellular DDR activation in many viruses.
In summary, the incoming viral genome, the activity of viral proteins, and viral DNA replication can serve as triggers for DDR activation. Recognizing virus-specific triggers and the mechanism involved in DDR activation is essential to a complete understanding of virus-DDR regulation. It would also be interesting to know whether these activation triggers work independently or in combination to induce cellular DDRs. As explained before, HPV E7 protein directly activates ATM response, which is then amplified upon viral DNA replication (Moody and Laimins, 2009). Similarly, initial DDR foci formation observed in a replication defective Ad mutant is only amplified in replication competent mutants (Chapter II). These findings suggest that viruses may provide multiple triggers for a full activation of cellular DDRs.
Requirements for G2/M cell cycle arrest in virus infection
Cells prevent mitotic progression and induce G2/M cell cycle arrest in the presence of DNA damage or incomplete replication. A variety of viruses are known to cause G2/M cell cycle arrest. Induction of G2/M cell cycle arrest provides a pseudo-S phase like environment where
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normal cellular DNA replication is complete, but the cell is still competent for replication (Chavy and Doorbar, 2007). Therefore, viruses use this arrest to their advantage to facilitate their replication. Although the mechanism of G2/M arrest is not fully characterized, it involves the regulation of cell cycle proteins such as Cdc2, Cyclin B1, and Cdc25c. These proteins are regulated either by the direct action of viral proteins or indirectly by cellular DDRs induced by viral proteins, presence of the viral genomes, and unusual viral DNA structures (reviewed in Davy and Doorbar, 2007).
G2/M cell cycle arrest is induced by the direct action of some viral proteins. The JC polyomavirus agnoprotein and the HPV E4 protein induce G2/M cell cycle arrest by inhibiting Cdc2/cyclin B1 kinase activity, which is required for pushing the cell into mitosis (Darbinyan et al., 2002; Knight et al., 2006). Indirectly, the HIV Vpr protein binds to cellular chromatin and activates ATR kinase activity (Lai et al., 2005). The ATR downstream kinase Chk1 inactivates Cdc25c phosphatase and induces G2/M cell cycle arrest (reviewed in Abraham, 2001). Adeno- associated virus DNA, which is single-stranded with hairpin structures at both ends, is sufficient to activate ATM-dependent signaling and G2/M cell cycle arrest (Raj et al., 2001). In another parvovirus, minute virus of mice, viral DNA replication correlates with ATM-dependent signaling (Adeyemi et al., 2010), suggesting that unusual viral structures generated during viral DNA replication (Cotmore and Tattersall, 1994) are likely a trigger for ATM signaling and G2/M cell cycle arrest (De Beeck and Caillet-Fauquet, 1997).
Ad E4 mutant 1007 activates and co-localizes ATM-and ATR-dependent proteins at viral replication centers (Stracker et al., 2002; Carson et al., 2003). E4 mutant infection causes G2/M cell cycle arrest (Cherubini et al., 2006). However the relationship between viral DNA replication and G2/M cell cycle arrest is unclear in Ad infection. We have observed that replication defective E1- does not induce G2/M cell cycle arrest, whereas replication competent E4 mutant 1007 does (Chapter II). This suggests that the initial focal accumulation of DDR proteins observed in E1- infections is not sufficient to activate cell cycle arrest. To our surprise, we observe that replication competent E4 mutant 1010 which was unable to maintain activated ATM dependent proteins at viral replication centers did not induce G2/M cell cycle arrest (Chapter II). This raises the possibility that activated ATM is not sufficient for G2/M cell cycle arrest, if not localized to viral DNA replication centers. However, Carson et al. (2009) have 87
observed that a mutant similar to 1010, which expresses E4-11kDa, inactivates ATR (Carson et al., 2009), so it is also possible that E4 mutant induced G2/M cell cycle arrest is ATR rather than ATM dependent. In conclusion, viral DNA replication mediated amplification of DDR signaling plays an important role in G2/M cell cycle arrest (Chapter II). We also find that E4-11kDa mediated expression prevents G2/M cell cycle arrest (Chapter II), but the mechanism is not yet determined, and requires further investigation.
Adenovirus as a model system for understanding DDR
We became interested in cellular DDR activation in Ad infection due to our previous observation that some aspects of the DDR such as focal accumulation of Mdc1 occur before the onset of viral DNA replication (Mathew and Bridge, 2007). Other aspects such as phosphorylation of Chk1 and p53 happen after viral DNA replication (Carson et al., 2003).These findings suggest that some DDR proteins are activated in response to incoming unreplicated viral genomes whereas others may require viral DNA replication. Using various Ad mutants, we have identified a role for incoming unreplicated viral genomes in DDR foci formation, and a role for viral DNA replication in amplification of cellular DDRs (Chapter I and II). Based upon my dissertation work and observations from other researchers, I propose the following model to explain the role of incoming viral genome and viral DNA replication in cellular DDR activation (Fig. 16).
We have observed focal accumulation of Mre11, pATM, Mdc1, γH2AX, pChk2, and 53BPI in Ad infection before the onset of viral DNA replication (Chapter I and II), suggesting the incoming viral genome triggers foci formation. As described above, transcription mediated chromatin remodeling of the Ad genome is required for pATM and Mdc1 foci formation (Karen and Hearing, 2011; Chapter I). We also observed that pATM foci co-localize with other DDR proteins (Chapter II). How are DDR foci induced in Ad infection? We have previously shown that Mre11 of MRN complex binds to the viral genome and Mre11 knockdown abrogates Mdc1 foci formation (Mathew and Bridge, 2008). Recent observations from our lab showed that Nbs1- cells that lack an intact MRN complex fail to induce pATM foci (Gautam and Bridge, 2013). Taken together, these findings suggest that the transcriptionally remodeled input viral genome is sensed by the MRN complex, leading to recruitment and downstream activation of ATM, which
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Figure 16: Model for Ad induced cellular DDR. See text for explanation.
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phosphorylates multiple effector molecules such as H2AX and Chk2. The transcriptionally remodeled viral genome, which induces focal accumulation of multiple DDR proteins, likely resembles broken cellular DNA. The focal accumulation of DDR proteins is observed in response to DSBs induced by various exogenous and endogenous agents (Pinder et al., 2013). The replication defective Ad mutant delivers incoming viral genomes which mimics DSBs induced by other DDR agents. Therefore, this replication defective mutant can serve as a model to understand DDRs induced as a result of DSBs, without the potentially complicating factor of concomitant DNA replication.
Previous findings suggest that Ad viral DNA replication contributes to DDR activation (Carson et al., 2003). We and others have observed that phosphorylation of ATM and its dependent substrates Nbs1, Chk2, and H2AX is amplified by the process of viral DNA replication rather than the increase in copy number as a result of replication (Chapter I and II; Nichols et al., 2009). How does Ad viral DNA replication amplify cellular DDR? Ad genome replication gives rise to single stranded (ss) DNA intermediates, ds DNA, and unusual replication intermediates such as pan-handle structures (Challberg and Kelly, 1989), all of which could serve as a potential trigger for ATM and ATR signaling pathways. During cellular DNA repair induced by other DDR agents, ssDNA coated with replication protein A (RPA) and junctions of ssDNA and dsDNA serve as triggers for ATR signaling (Zou 2007). Similarly, in replication-competent E4 mutant infections, ssDNA and RPA accumulate at viral replication centers and activate ATR- dependent signaling (Pombo et al., 1994; Carson et al., 2009). This suggests the possibility that ssDNA and other replication intermediates may amplify DDR activation of ATM and/or ATR pathways, or that communication between ATM and ATR pathways contributes to full DDR activation.
There are distinct parallels in DDR activation by virus infection and other DNA damaging agents. However, DDR activation by other agents is complicated by a multitude of factors such as type of agents, chromatin organization, status of transcription and replication, and chromatin modifications (reviewed in Polo and Jackson, 2011). In contrast, use of Ad mutants with limited gene expression and replication capabilities provides a unique opportunity to investigate the role of the incoming genome, as well as processes such as genome transcription and replication, in DDR activation. 91
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