UNIVERSITY OF CALIFORNIA, SAN DIEGO
DNA Viruses: Pathogens, Therapies and Compasses for Navigating Cancer
A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy
in
Biology
by
Kristen Carolyn Espantman
Committee in charge:
Professor Clodagh O’Shea, Chair Professor Ghorisankar Ghosh Professor Steven Wasserman Professor John Young Professor Elina Zuniga
2014
The Dissertation of Kristen Carolyn Espantman is approved, and it is acceptable in quality and form for publication on microfilm and electronically:
Chair
University of California, San Diego
2014
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DEDICATION
This dissertation is dedicated to everyone who had no doubt that I would finish my PhD successfully. My parents have been extremely supportive for my entire life and have allowed me to follow my instincts. They have set amazing examples of what it means to be a hard-working and honorable individual. Through them I have gained support through my many family members: brothers, grandparents, aunts, uncles and cousins. I am a lucky person to have always been surrounded by people who believe in me and love me. Two people who kept me sane during graduate school were my best girlfriend Christine and my favorite lab-mate Conrado. They were there for me every time things got tough and kept me smiling. I was the first person to join the O’Shea lab. Since then the lab has grown into a family and I dedicate this achievement to every one of them. Our fearless leader Clodagh is an inspirational scientist who has molded me into a great scientist. She has transformed me from a biology novice to a confident experimentalist. This dissertation is also dedicated to all future graduate students that will graduate from the O’Shea lab. Lastly, I would like to dedicate this to my husband and best friend Mike. Mike says that I am the most competent person that he knows. He has endless confidence in me and my abilities. Our mutual respect for one another has driven us both to be the best people we can be. I have also gained a new family through Mike who are also extremely supportive and loving. Thank you everyone for enriching my life.
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EPIGRAPH
“The reward for work well done is the opportunity to do more.” –Jonas Salk
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TABLE OF CONTENTS
Signature Page………………………………………………………………………… iii
Dedication……………………………………………………………………………… iv
Epigraph………………………………………………………………………………... v
Table of Contents……………………………………………………………………… vi
List of Figures……………………………………………………………………….…. viii
List of Tables…………………………………………………………………………... x
Acknowledgements………………………………………………………...…………. xi
Vita………………………………………………………………………………………. xii
Abstract of the Dissertation…………………………………………………………... xiii
Introduction……………………………………………………………………….…….. 1
Chapter 1. Heterochromatin silencing of p53 target genes by a small viral protein ……………..………………………………………………………………….. 20
Abstract……………………………………………………………………..…………. 20 Introduction/Results……………………………………………………………….….. 20 Discussion……………………………………………………………………………... 35 Materials and Methods……………………………………………………………….. 39 Author Contributions…………………………………………………………………… 43
Chapter 2. p53 inactivation by oligomeric E4-ORF3 is specific to seroprevalent subgroup C adenoviruses ……………………….…….…….….. 44
Abstract……………………………………………………………………..………….. 44 Introduction ……………………………………………………………….…………... 45 Results……….……………………………………………………………..………….. 49 Discussion……………………………………………………………………………... 68 Materials and Methods……………………………………………………………….. 64 Author Contributions…………………………………………………………………… 71
Chapter 3. The TRIM family is targeted by multiple oligomeric DNA virus proteins known to disrupt PML ………………………………………………….. 72
Abstract……………………………………………………………………..………….. 72 Introduction………. ……………………………………………………………….….. 73 Results……….……………………………………………………………..………….. 75 Discussion……………………………………………………………………………... 100 Materials and Methods……………………………………………………………….. 105 Author Contributions…………………………………………………………………… 107
Conclusions and Final Remarks…………………………………………………….. 108
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Appendix………………………………………………………………………………… 111 References……………………………………………………………………………… 213
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LIST OF FIGURES
Introduction
Figure 1.0 Human TRIM proteins………………………………………………… 13
Chapter 1 Main Figures
Figure 1.1 p53 is induced and phosphorylated in ∆E1B-55k infection but p53 activity is dominantly suppressed…………………………………………………. 25
Figure 1.2 E4-ORF3 inactivates p53 independently of E1B-55k and p53 degradation…………………………………………………………………. 27
Figure 1.3 E4-ORF3 induces heterochromatin formation and prevents p53-DNA binding at endogenous promoters………………………………………………… 29
Figure 1.4 E4-ORF3 forms a nuclear scaffold that specifies heterochromatin assembly at H3K9 trimethylation at p53 target promoters………………………. . 32
Figure 1.5 Transient knockdown of SUV39H1 and SUV39H2 is not sufficient to decrease H3K9me3 levels and prevent E4-ORF3 induced silencing of p53 transcriptional targets……………………………………………………… 34
Figure 1.6 p53 transcriptional targets are silenced selectively in the backdrop of global transcriptional changes that drive oncogenic cellular and viral replication 36
Chapter 2 Main Figures
Figure 2.1 - Subgroup C E4-ORF3 protein sequences diverge from all other adenovirus subgroups. ……………………………..…………………………………… 51
Figure 2.2 - Engineering and validation of ∆E1B-55K and ∆E1B-55K/∆E4-ORF3 mutant adenoviruses. ………………………………………………………………. 54
Figure 2.3 - Engineering and validation of ∆E1B-55K mutant adenovirus with Ad9, Ad12 or Ad34 E4-ORF3 genes replacing E4-ORF3 from Ad5……………….. 55
Figure 2.4 - Ad9, Ad12 and Ad34 E4-ORF3 proteins do not suppress activation of p53 transcriptional targets or support viral replication.…………..… ………. 57
Figure 2.5 - Mislocalization of PML, TRIM24 and TRIM33 is a conserved function of E4-ORF3 among disparate adenovirus subgroups and is not sufficient to inactivate p53 target genes. …………………………………….……………. 59
Figure 2.6 - The MRN complex is only mislocalized in cells infected with viruses expressing Ad5 E4-ORF3 that are able to replicate and inactivate p53. ……….…… 60
Figure 2.7 – Residues of the β-barrel interface and solvent exposed residues of E4-ORF3 dimer are most different in comparison of E4-ORF3 amino acid sequences from disparate serotypes. ………………………………………………………….. 63
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Figure 2.8 - The crystal structure of E4-ORF3-Ad5 reveals the similarities and differences among disparate E4-ORF3 proteins. .……………………………………… 64
Chapter 3 Main Figures
Figure 3.1 – The family of TRIM proteins form a network based on protein-protein interactions. ……………………………….………………………….… 77
Figure 3.2 – TRIM proteins interact with each other via multiple domains…….… 79
Figure 3.3 – The network of TRIMs and first order interacting proteins reveal their likely functions in a wide array of cellular processes and pathways. 81
Figure 3.4 – Schematic of high throughput screening to determine targets of E4-ORF3…. ……………………………………………………………… 83
Figure 3.5 – E4-ORF3 mislocalizes a subset of the TRIM family in the nucleus or cytoplasm …………………………….………………….……………….. 84
Figure 3.6 – MYC tagged E4-ORF3 accumulates in the nucleus and cytoplasm revealing additional potential TRIM targets.………..…………….….. 85
Figure 3.7 – ICP0 mislocalizes a subset of the TRIM family…………………. … 89
Figure 3.8 – ICP0 causes degradation of a subset of TRIM proteins …...... 90
Figure 3.9 – Venn diagram of TRIMs revealed to interact with E4-ORF3 and ICP0 . … 91
Figure 3.10 – E4-ORF3 does not target a single TRIM domain.…………………….. 93
Figure 3.11 – ICP0 mislocalizes TRIM proteins via multiple domains within each TRIM… 95
Figure 3.12 – Super-resolution images of TRIMs with E4-ORF3 reveals details of TRIM-E4-ORF3 interactions…………………………………………… 97
Figure 3.13 – Super-resolution images of TRIMs with ∆RING-ICP0 reveals details of TRIM-ICP0 interactions.……… …………………………………………. 99
Figure 3.14 – IE2 interacts with a subset of the TRIM family in diverse nuclear structures 101
Figure 3.15 – Venn diagram of TRIMs revealed to interact with E4-ORF3, ICP0 and IE2 102
Chapter 3 Supplementary Figures
Supplementary Figure 3.1 – Interaction Results of TRIM domain mutants co-expressed with full length TRIMs or viral proteins …………………… 118
Supplementary Figure 3.2 – Interaction Results of FLAG-TRIMs co-expressed with disparate viral proteins …………………………..………… 147
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LIST OF TABLES
Chapter 2 Supplementary Tables
Supplementary Table 2.1 - List of Adenovirus protein IDs from NCBI used in this study 111
Supplementary Table 2.2 - Calculations of Surface Exposed Residues of E4-ORF3 as determined by the crystal structure of N82 E4-ORF3 from Ad5 113
Chapter 3 Supplementary Tables
Supplementary Table 3.1 Degrees of protein-protein interactions within the network of the TRIM family of proteins and their first order interactors.. 193
Supplementary Table 3.2 Top Gene Ontology (GO) Biological Processes significantly overrepresented by the protein network of first order TRIM interacting proteins……………………………...... 200
Supplementary Table 3.3 Top canonical signal transduction pathways significantly overrepresented by the protein network of first order TRIM interacting proteins………….……………………………….. 206
Supplementary Table 3.4 Tripartite Motif proteins (TRIMs) used in this study……. 212
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ACKNOWLEDGEMENTS
Conrado Soria and Dr. Fanny Estermann provided the data to support the major
conclusions Chapter 1. Led by Dr. Clodagh O’Shea we were able to work as a team to complete
and publish this as a manuscript in August, 2010 in Nature called “Heterochromatin Silencing of
p53 target genes by a small viral protein”. We thank Dr James Fitzpatrick and the Waitt
Advanced Biophotonics Center for assistance with data acquisition and image analysis. We thank
Drs Jan Karlseder, Inder Verma, Tony Hunter, Reuben Shaw and members of the O’Shea
laboratory for critical reading of this manuscript. We acknowledge Drs Luciano Haro and Satchin
Panda for their help in setting up Real-Time p53-luciferase assays. In addition, we are very
grateful to Drs. Joaquin Espinosa and Roddy O’Sullivan for protocols and advice in performing
chromatin immunoprecipitation experiments. We thank Dr. Phil Branton and Dr. David Ornelles
for the kind gift of adenovirus mutants. C.C.O.S would like to acknowledge funding from the
Alliance of Cancer Gene Therapy, the American Cancer Society, the Sontag Foundation and the
Arnold and Mabel Beckman Foundation .
I thank Dr. Jason DeHart of the O’Shea lab for processing and analyzing RNA expression
levels using his expertise in qPCR in Chapter 2. I would like to thank Dr. Colin Powers of the
O’Shea lab for developing a straightforward and fast method for engineering adenovirus mutants
that allowed me to quickly create and validate E4-ORF3 adenovirus mutants used in this study. I
thank Dr. Horng Der Ou for the analysis of E4-ORF3 solvent exposed residues and his constant
advice regarding E4-ORF3 structure and function.
I thank Dr. Clodagh O’Shea for being a driving force behind the project in Chapter 3 and
supporting my conviction in using the entire family of TRIM proteins throughout this work. I also
thank Erik Kapernick for his valuable help using the ImageXpress microscope and software.
xi
VITA
2003 Bachelor of Science, University of California, Los Angeles
2003 Intern in Process Development, Amgen, Thousand Oaks, CA
2004 Research Associate I in Pharmaceutics, Amgen, Thousand Oaks, CA
2004-2006 Research Associate in Process Development, Amgen, Thousand Oaks, CA
2007-2008 Teaching Assistant, Department of Biological Sciences, University of California,
San Diego
2014 Doctor of Philosophy in Biology, University of California, San Diego
PUBLICATIONS
Espantman KC, O’Shea CC. “The TRIM family is targeted by multiple oligomeric DNA virus proteins known to disrupt PML,” in progress
Espantman KC, Dehart J, O’Shea CC. “p53 inactivation by oligomeric E4-ORF3 is specific to seroprevalent subgroup C adenoviruses,” submitted to JVI 2014
Soria C., Estermann FE, Espantman, KC, O’Shea CC. “Heterochromatin silencing of p53 target genes by a small viral protein,” Nature 466 (7310):1076-81 (2010)
Espantman KC, O’Shea CC. “aMAGEing new players enter the RING to promote ubiquitylation,”
Molecular Cell 39(6):963-74 (2010)
xii
ABSTRACT OF THE DISSERTATION
DNA Viruses: Pathogens, Therapies and Compasses for Navigating Cancer
by
Kristen Carolyn Espantman
Doctor of Philosophy in Biological Sciences
University of California, San Diego, 2014
Professor Clodagh O’Shea, Chair
The study of DNA virus-host protein interactions has revealed cellular proteins critical for cell growth and survival. In addition, the adenovirus has potential as a potent cancer therapy against the majority of tumors which lack a functional p53 pathway. For this oncolytic adenovirus to be realized, the ability of adenovirus to inactivate the p53 pathway must be eliminated. In
Chapter 1, we reveal that a small early adenovirus protein E4-ORF3 prevents p53 from accessing
DNA by instigating de novo heterochromatin formation specifically at p53 target genes.
xiii
Unfortunately, full deletion of E4-ORF3 and another adenovirus protein that inactivates p53, E1B-
55K, renders the virus defective for replication, even in cells lacking p53 function. Therefore, it is imperative to reveal the critical functions and residues of E4-ORF3 necessary to block p53 activation and support viral replication. In Chapter 2, we show that among the 7 diverse adenovirus subgroups, only Subgroup C E4-ORF3 proteins are capable of supporting viral replication and inactivating p53 target genes in an E1B-55K deleted adenovirus. Furthermore, we reveal that MRN mislocalization is also specific to Subgroup C E4-ORF3 while targeting of PML,
TRIM24 and TRIM33 is conserved across subgroups. Thus, comparison of E4-ORF3 protein sequences from disparate subgroups reveals residues likely to be required for p53 target gene inactivation leading to creation of an oncolytic adenovirus in which this function is removed.
Conserved E4-ORF3 targets PML, TRIM24 and TRIM33 are members of the superfamily
of Tripartite Motif proteins (TRIMs) that have similar oligomeric N-terminal domain structures and
varied C-termini. E4-ORF3 may be targeting a critical subset of TRIM proteins for the benefit of
adenovirus replication. In Chapter 3, we show that the TRIM interactome including first order
interacting proteins contains over 900 nodes and 12,500 edges with members regulating a
plethora of cellular pathways disrupted during virus replication. Using high-throughput imaging,
we reveal 21 additional TRIMs that interact with E4-ORF3 and show that disparate PML targeting
viral proteins Herpes Simplex Virus-1 ICP0 and Cytomegalovirus IE2 also disrupt 18 or 7 TRIMs,
respectively. Moreover, we demonstrate that these viral proteins do not target a common TRIM domain, and that oligomerization among TRIMs can contribute to TRIM-viral protein interactions.
Taken together, this work reveals novel functions of DNA virus proteins that will contribute to anti-
viral and anti-cancer therapies to facilitate the human pursuit of life and happiness.
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Introduction
Small DNA tumor viruses: research and discoveries
Small DNA tumor viruses have evolved proteins that target the critical cellular pathways that regulate cell growth and survival. The discovery of mouse polyoma virus in the 1950’s opened the door for researchers to explore the role of DNA viruses as cancer-inducing agents which lead to the discovery of numerous significant insights into the mechanisms underlying human cancer [1]. The group of small DNA tumor viruses include polyomaviruses, adenoviruses and the papillomaviruses; some herpesviruses (Epstein-Barr Virus and Kaposi’s Sarcoma
Hepresvirus) and the hepadnaviruses (Hepatitis B virus) are also associated with human cancers
[2]. For these DNA viruses to effectively reproduce, they must first initiate cellular DNA replication, while simultaneously blocking apoptosis and damping the body’s immune system. To achieve this host cell take over, DNA viruses encode proteins that manipulate host cell proteins and DNA in order to turn on and off the desired pathways [3].
The study of proteins encoded by small DNA tumor viruses have led to the discovery of fundamental cellular processes such as mRNA splicing, polyadenylation signals, protein nuclear localization signals, ubiquitin-dependent degradation, and the identification of the first known E3 ubiquitin ligase [4-8]. In addition, major players involved in cellular transformation were revealed by their binding to small DNA tumor virus proteins. The retinoblastoma gene product, pRB, was first found as a cellular protein bound to the adenovirus oncoprotein E1A [9]. Moreover, p53 was first detected as a protein bound tightly to SV40 large T-antigen, however the significance of this interaction was not appreciated for nearly ten years after its discovery when p53 was identified as a tumor suppressor gene whose function is compromised by large T-antigen [10-12]. One of the most striking similarities among the DNA tumor viruses is their functional overlap of cellular targets. pRB and p53 are not only targeted by SV40 and adenovirus, respectively, but they are both targeted by all small DNA tumor viruses. Specifically, p53 is targeted by SV40 large T- antigen, adenovirus E1B-55K, and human papillomavirus E6 [8, 13] and pRB is targeted by SV40 large T-antigen, adenovirus E1A and human papillomavirus E7 [14, 15]. Intriguingly, the viral
1 2
proteins that target pRB have evolved a common LxCxE motif that binds directly to a conserved
“pocket” region of the family of retinoblastoma proteins, pRb, p107 and p130. These functional overlaps observed among disparate viral proteins illustrate the importance of p53 and the RB family of proteins in regulating cellular growth and survival and have facilitated the field of cancer research in narrowing the key players involved in cancer initiation, progression and survival.
Furthermore, research of small DNA tumor virus proteins also revealed that certain oncogene
products cooperate with one another in eliciting a transformed phenotype. This observation
emerged initially from studies with adenovirus showing that both E1A and E1B were required for
cellular transformation, illustrating the need to initiate DNA replication, and also inhibit
downstream blockades [16]. Consequently, the field of cancer biology has benefitted greatly from
the discoveries uncovered from exploring the functions and targets of small DNA tumor viruses
and will continue to benefit as more targets are revealed.
p53 and Cancer
p53 is considered the “Guardian of the Genome” as its role is to preserve genomic
stability, responding to a variety of insults including DNA damage, hypoxia, metabolic stress, loss
of cell-cell adhesions, and oncogene activation [17]. It is a transcription factor that elicits its
effects on the cell by activating transcription of many genes that govern the major defenses
against tumor growth [18]. A dividing cell lacking a functional p53 pathway will acquire aberrant
DNA damage potentially resulting in the activation of oncogenes or loss of additional tumor
suppressors leading to the development of a metastatic cancer. The progression of cancer is
very complex, with over 100 distinct types of cancer stemming from the many tissues of the
human body, however, mutations that result in the loss of p53 transcriptional activation are a
common feature of almost all cancers [19].
Given that p53 function is critical for growth regulation and is lost in the majority of
cancers, efforts have been made to develop cancer treatments that attempt to re-activate the p53
pathway. Unfortunately, these methods have been largely unsuccessful due to the lack of cellular
targets and imprecise methods of “drug” delivery. Nutlin is one such drug developed to re- 3
activate p53 by stabilizing p53 protein levels [20]. It does so by blocking the interaction between
MDM2, a potent E3 ubiquitin ligase, and its substrate, p53 [21]. Unfortunately, this treatment is extremely limited as a cancer treatment since the cancer cells must express wild-type p53 for
Nutlin to be effective [22, 23]. Furthermore, during treatment with Nutlin, the cancer may adapt, acquiring further mutations to inactivate p53 and eventually evade the treatment rendering Nutlin ineffective. Another popular method being developed as a cancer treatment is to re-introduce wild-type p53 into tumor cells using gene therapy [24]. There are a few major caveats to this method, one being the method of delivery. To re-introduce wile-type p53 into a cell, an active p53 gene needs to be delivered to the nucleus. To do this, scientists have engineered replication incompetent viruses carrying a wild-type p53 gene [25]. The virus would need to infect the cancer cells, insert the p53 gene into the nucleus, and express the p53 protein. Unfortunately, it would be virtually impossible for all of the cancer cells in a massive tumor to be infected with the virus, and furthermore, due to the high frequency of DNA mutations in tumors, including mutations in p53 leading to a dominant-negative phenotype, re-introduction of wild-type p53 alone is insufficient [26]. All things considered, p53 is still an extremely attractive target for novel cancer therapies, but how does one target something which is lost?
Adenovirus proteins and replication
Much like cancerous cells, quiescent cells infected with adenovirus are forced into S phase, driving DNA replication, while inactivating tumor suppressors to evade cell cycle arrest and apoptosis [3]. To achieve this initiation of cell cycle, the adenovirus encodes proteins that have evolved efficient strategies of manipulating the cellular program. The adenovirus’ 36kb genome encodes genes that are divided into early (E) and late (L) stage transcriptional elements to advance replication. The late proteins are required for viral assembly and packaging, while the early proteins commandeer cellular factors to initiate and progress viral genome replication.
There are approximately twenty early adenovirus proteins that work in concert, transforming the host cell into a virus production factory. Adenovirus E1A is the first gene to be transcribed and its protein products play a major role in the transcriptional regulation of viral and cellular genes [27]. 4
E1A binds to RB, blocking the RB-checkpoint and inducing E2F activity [9]. However, forced entry into S-phase induced by E2F activity provokes p53-dependent apoptosis [28].
Consequently, E1B-55K and its viral binding partner E4-ORF6 jointly mediate p53 degradation through ubiquitin mediated proteolysis [29]. E1B-55K has E3 ubiquitin ligase activity and directly binds to the p53 transactivation domain, blocking p53 transcriptional activity and also exports p53 to the cytoplasm while E4-ORF6 recruits host cell ubiquitinylation machinery such as Cul5, Rbx1 and elongins B and C to induce p53 polyubiquitinylation [30-33]. E2F activation coupled with p53 inhibition is a combination widely observed in cancers. Consequently, expression of E1A and
E1B are sufficient to transform quiescent cells [16].
At later times of infection, late viral protein translation is promoted and viral DNA forms nuclear replication domains coated with the adenovirus E2A protein. Viral capsid assembly and genome packaging occurs in the host cell nucleus starting 20 to 24 hours post infection. Two to three days post infection, the cells begin to lyse and release virions. Virus release is aided by the expression of the adenovirus death protein (ADP), a nuclear membrane glycoprotein encoded by the E3 region. Over-expression of ADP during viral infection has been shown to enhance cell lysis and more effectively release virion particles from the host cell [34]. The adenovirus is an effective killer of human cells, and as such, is the perfect platform for which to engineer a targeted therapy to selectively kill tumor cells. p53 selective tumor therapy
The similarities between adenovirus infected cells and tumor cells are quite striking. In fact, the environment inside a tumor cell would allow certain mutant adenoviruses to replicate, where in normal cells, replication would be abrogated. For example, an adenovirus lacking the
E1B-55K gene, whose function is to inactivate p53, would be complemented for replication in a cancer cells with an inactivated p53 pathway. This premise was the basis for an oncolytic adenovirus coined ONYX-015. ONYX-015, also known as dl1520, is an adenovirus that lacks the ability to express E1B-55K. Cells infected with ONYX-015 induce high levels of p53 which was expected to limit viral replication in normal cells, but not p53 mutant tumors. On this basis, 5
ONYX-015 was tested in patients as a p53 tumor-selective oncolytic viral therapy and is now approved in several countries (known as Oncorine). However, the loss of E1B-55K functions in viral RNA export, rather than p53 inactivation, is the major determinant of ONYX-015 tumor selectivity [35, 36]. Contrary to expectations, although p53 accumulates to high levels in the nucleus of ONYX-015 infected normal cells, p53 transcriptional targets are not induced [36]. The failure of p53 stabilization to activate transcriptional targets is not a tissue-specific effect and occurs in multiple primary cell types and tumor cell lines. This revealed a fundamental gap in our understanding of not only adenovirus biology, but also p53 activation as it was previously believed that high p53 levels were tantamount to its transcriptional activation. Thus, the potential for a p53 selective viral therapy remains to be realized.
Adenovirus E4-ORF3
The lack of transcriptional activation of p53 target genes during ONYX-015 was a mystery given that the E1B-55K protein was not expressed, and high levels of p53 protein were allowed to accumulate during infection. Chapter 1 reveals that another adenovirus protein, E4-
ORF3, blocks transcriptional activation of p53 target genes and that only when E1B-55K and E4-
ORF3 are both deleted are p53 target genes turned on during viral replication [37]. These results indicated that E4-ORF3 was responsible for the inactivation of p53 target genes, however, the mutant virus lacking E1B-55K and E4-ORF3 expression used in this discovery also lacked expression of another adenovirus early protein E4-ORF3-4. Furthermore, it was unclear if other adenovirus early proteins were necessary for E4-ORF3 to block p53 transcriptionally activated genes. Therefore, I ectopically expressed E4-ORF3 in cells infected with a mutant adenovirus lacking expression of E1B-55K and E1A-13s (∆55K/∆13s), required for activation of the adenovirus E4 genes, and revealed that E4-ORF3 complements ∆55K/∆13s for p53 target gene
inactivation when compared to the control. These data revealed that E4-ORF3 alone was
responsible for the lack of transcription from p53 target genes even though high levels of p53
were present during ONYX-015 infection. 6
Chapter 1 also reveals the mechanism by which E4-ORF3 silences p53 mediated genes.
Initially, we hypothesized that p53 was post-translationally modified in an inactivate state or lacking an interaction with a critical transcriptional co-activator that rendered p53 unable to activate transcription. However, my work revealed that p53 was able to activate transcription of an ectopic luciferase plasmid under the control of p53 binding sites, revealing that, during infection with ONYX-015, p53 was in a transcriptionally activate state. This finding revealed that
E4-ORF3 likely had a role in modifying the cellular DNA, as one major difference between ectopic and cellular DNA is the presence of histones on chromatin that can serve as a blockade to transcriptional activation. Remarkably, research performed by Dr. Fanny Estermann found that,
E4-ORF3 silences p53 target genes by inducing de novo heterochromatin formation specifically at genes transcriptionally activated by p53. E4-ORF3 is a polymer that oligomerizes in the nucleus of infected cells forming irregular fibers that co-localize with Histone H3 Lysine 9 trimethyl
(H3K9me3), a signature of heterochromatin. E4-ORF3 simultaneously co-localizes with histone methyltransferases SUV39H1 and 2. We hypothesized that E4-ORF3 requires SUV39H1 and 2 to induce H3K9me3, however, SUV39H1 and 2 knockdowns performed by Conrado Soria and myself, showed that neither were required for E4-ORF3 to inactivate p53 target genes. Taken together, Chapter 1 reveals a novel mechanism for inactivation of the p53 pathway through adenovirus E4-ORF3 and gives us a starting place to build a truly p53 selective oncolytic adenovirus.
E4-ORF3 is a 13kDa protein that weaves through the nucleus of infected cells forming irregular fibers that polymerize via reciprocal and non-reciprocal swapping of E4-ORF3 C-terminal tails [38]. The mechanism by which E4-ORF3 oligomerizes was discovered by Dr. Horng Ou, a post-doc in the O’Shea lab. His work also revealed that E4-ORF3 functions by forming an avidity surface whereby this small protein forms a dominant protein interaction matrix to capture and disrupt multiple large cellular protein complexes including known E4-ORF3 targets the DNA damage complex composed of Mre11, Nbs1 and Rad50 (MRN complex) [39], and the oligomeric proteins Promyelocytic Leukemia (PML) [40], TRIM24 [41], and TRIM33 [42]. Dr. Ou also 7
showed that the oligomerization of E4-ORF3 into nuclear fibers is necessary to mislocalize its cellular protein targets and block activation of p53 target genes. To engineer a truly p53 selective oncolytic adenovirus, it is critical to know which functions of E4-ORF3 are necessary for effective adenovirus replication, and if those functions can be separated from E4-ORF3’s ability to silence p53 target genes.
Adenovirus type 5 (Ad5) from Subgroup C is the most widely studied adenovirus due to
its seroprevalence and use in viral vectors, however there are 68 distinct adenoviruses within 7
disparate subgroups [43, 44]. E4-ORF3 from Ad5 disrupts MRN, PML, TRIM24, TRIM33 and p53
simultaneously, though it has not been tested if disparate E4-ORF3 proteins from additional
subgroups are functionally conserved. In Chapter 2, I show that hybrid Ad5 adenoviruses, in
which E1B-55K is deleted and the E4-ORF3 gene of Ad5 is replaced with that from Ad9,
Subgroup D, Ad12, Subgroup A or Ad34, Subgroup B, do not inactivate p53 target genes,
revealing that this function is unique to E4-ORF3 from Subgroup C. However, Ad9, 12 and 34
E4-ORF3 proteins expressed from the hybrid viruses do have conserved functions with Ad5 E4-
ORF3 in mislocalizing PML, TRIM24 and TRIM33, Illustrating that mislocalization of these cellular
proteins are not sufficient to block p53 target gene inactivation. The data in Chapter 2 also reveal
that along with its function in p53 target gene inactivation, E4-ORF3 from Subgroup C is unique in
its ability to mislocalize the MRN complex and support viral replication in Ad5 ∆E1B-55K. This
suggests that mislocalization of the MRN complex and p53 target gene inactivation are somehow
linked. We hypothesize that Subgroup C E4-ORF3 proteins have evolved a novel interaction
interface defined by non-conserved surface residues that give rise to additional protein
interactions and lead to its unique functions. Using the crystal structure of the Ad5 E4-ORF3
soluble dimer mutant [38], we define solvent exposed residues and suggest residues likely to
contribute to both conserved and non-conserved functions of E4-ORF3 proteins. The discovery
that E4-ORF3 proteins from Subgroup A, B and D have natural mutations to separate their ability
to mislocalize PML, TRIM24 and 33 with the ability to inactivate p53 target genes will further
support the construction of a potent p53 selective oncolytic adenovirus. 8
The Promyelocytic Leukemia protein (PML)
PML was first discovered in Acute Promyelocytic Leukemia (APL) where due to a chromosomal translocation, the N-terminus of PML is fused to the DNA and retinoic acid (RA) binding domains of the Retinoic Acid Receptor-α (RARα) [45]. The PML-RARα fusion protein misregulates the transcription of many genes by recruiting corepressors and histone deacetylases to RA-target genes, thus acting as a dominant-negative RARα mutant [46].
Furthermore, the PML-RARα fusion protein disrupts PML bodies into microspeckled nuclear structures via homodimerization with wild type PML [47]. Repression of RA target genes combined with PML body disruption leads to a blockage in differentiation and uncontrolled replication of myeloid precursor cells [48]. Intriguingly, treatment of RA on primary cultures of bone marrow from patients with APL induces a progressive differentiation of the leukemic cells into mature granulocytes [49-51]. This observation has been successfully transposed in vivo where administration of oral all-trans RA can induce complete remission in virtually every APL patient [51, 52]. Upon treatment with RA, the PML-RARα fusion protein is degraded and PML bodies re-form. The contribution of PML disruption in the oncogenic role of leukemia development is believed to mostly be through PML’s functions in regulating apoptosis where the absence of functional PML inhibits apoptosis and gives cells a selective growth advantage [53].
However, PML loss alone does not induce leukemia in knock-out mice, and furthermore, the mechanism by which PML functions is not clearly understood.
PML is also targeted by several DNA viruses which encode proteins that disrupt PML bodies using disparate mechanisms. Adenovirus E4-ORF3 mislocalizes PML from nuclear bodies onto E4-ORF3 nuclear cables, while Herpes Simplex Virus-1 (HSV-1) ICP0 binds and causes degradation of PML [54] . Cytomegalovirus (CMV) IE1 drives de-sumoylation of PML and related protein IE2 localizes adjacent to PML bodies early during CMV infection where it initiates viral replication domains [55]. The convergence of PML targeting by viral proteins and tumor mutations led scientists to believe that PML was a key regulator of cell growth and survival, as was true for checkpoint proteins p53 and Rb which are also converged upon by tumor mutations 9
and viruses. Decades of research, however, have shown that loss of PML function does not cause tumors, nor is PML a critical checkpoint protein [56, 57]. Why then do viral proteins converge in targeting PML? It is often overlooked that PML belongs to the TRIM super-family where it homo-oligomerizes and can hetero-oligomerize with multiple other TRIMs. We hypothesized that PML is not the central TRIM target of viral proteins, and instead propose that oligomeric viral proteins that target PML are actually targeting the TRIM family network. In
Chapter 3, I show evidence to support this hypothesis, and reveal a subset of TRIM proteins targeted by multiple DNA virus proteins.
The Tripartite Motif (TRIM) Family
Viruses and their hosts have co-evolved in the struggle for survival, both usurping the defensive strategies of the other. The host organism must acquire mechanisms to detect and destroy the foreign invader without destroying itself, whilst viral proteins mimic cellular structures, pirating the cell’s natural machinery for use in its own replication. Recognition of self, versus foreign proteins, presents an obstacle for the host organism’s innate immune system, the first line of defense against viral replication and spread. One defining feature of many viral proteins is their ability to form oligomers [58]. Recently, the TRIM family of proteins has been revealed as having a role in innate immunity where numerous TRIMs have been shown to restrict viral replication and are regulated by Interferons [59-62]. Most striking about this family is that virtually every domain within TRIM proteins can serve as a site for protein-protein interactions driving homo- and hetero-oligomerization within the TRIM family [63]. However, the significance of
TRIM-TRIM interactions among family members is unclear.
TRIM domain structure
The TRIM family consists of over 70 human members with broad functionality [64, 65].
The defining feature of the TRIM family is the conserved spacing of the domains at the N- terminus, composed of a RING (Really Interesting New Gene) domain, one or two B-box motifs and a coiled-coil (collectively called the RBCC domain). The RING domain chelates zinc via cysteine and histidine residues forming a structured domain often possessing intrinsic E3 10
ubiquitin ligase activity, in that it can promote the transfer of ubiquitin molecules from an E2 conjugating enzyme directly to lysine residues of target substrates [66]. Generally, poly-ubiquitin chains trigger protein degradation, while monoubiquitylation modulates target protein subcellular localization or function. Non-catalytic RING domains lacking ubiquitin ligase activity have been shown to stabilize the ubiquitin ligase activities of other proteins. One example of this is seen in p53 regulating proteins MDM2 and MDMX where the RING domain of MDMX does not possess ubiquitin ligase activity, and instead, stabilizes oligomeric conformations of MDM2 and increases the ubiquitin ligase activity of the RING domain of MDM2 instigating degradation of p53 [67].
Several TRIMs have been shown to have ubiquitin ligase activity resulting in protein degradation, such as TRIM32, TRIM25 and TRIM33 that mediate degradation of PIASY, RIG-I, and SMAD4, respectively [68-70], while it is unknown if any TRIM possesses a catalytically inactive RING domain.
TRIM proteins have one or two B-box motifs, named B-box1 and B-box2, always located between the RING and coiled-coil domains. The two types of B-box domains are cysteine- and histidine-rich, each with distinct zinc-binding consensus sequences and sizes, but with similar structures. B-box motifs have not yet been shown to have any ligase activity, although there is evidence of a role in enhancing the activities of RING domains [71]. The crystal structure of tandem B-box domains of MID1 (TRIM18) have been elucidated showing that the B-box motifs have an inter-molecular interaction reminiscent of RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box domains in regulating functional RING-type folds [71].
Within the TRIM family, B-box domains are consistently followed by a well known oligomerization motif, the coiled coil. The coiled coil was first described as the main structural element of a large class of fibrous proteins that included keratin, myosin and fibrinogen [72].
Subsequently, the coiled coil was found not only in fibrous proteins, but also in dimers and trimers serving as a homo-oligomerization motif. Coiled coils exist as bundles of α-helices from two or more proteins wound into a superhelix running in parallel or anti-parallel directions [73]. The hallmark of coiled coils is the distinctive packing of amino acid side chains in the core of the 11
bundle. Therefore, the ability of proteins to interact via their coiled coils is determined by extremely specific spacing of coordinating amino acids. Among the TRIM family, coiled coils serve as homo- and hetero-interaction motifs [74]. Together, the RBCC domain of TRIM proteins gives this family the ability to form higher-order homo- and hetero-oligomers and allow the TRIMs to form an extensive interactome [63] (and illustrated in Chapter 3).
The C-termini of TRIM proteins vary among the family providing them with diverse functions and separate the TRIMs into eleven sub-families (Figure 1.0 adapted from [62]) [60, 75].
Two-thirds of TRIM proteins possess a C-terminal SPRY or PRY-SPRY domain (Groups I and
IV). The PRY-SPRY domain serves as yet another protein-protein interaction motif and can form dimers or interact with other cellular proteins [76]. PRY-SPRY domain containing proteins are involved in innate immune and cytokine signaling and the domain is essential for the anti-viral functions of some TRIM proteins [60, 77]. One example is TRIM5α from Old World monkeys which interacts with the incoming HIV-1 viral capsid polyprotein Gag and promotes its premature uncoating and disassembly [78]. Conversely, the TRIM5α protein encoded by humans cannot interact with HIV-1 Gag and does not inhibit HIV-1 infection. Intriguingly, a single amino acid change in the SPRY domain of TRIM5α from Old World monkeys to humans gives this difference in protection from HIV-1 infection by eliminating human TRIM5α’s interaction with HIV-1 Gag [79].
Further studies show that the RBCC domain of TRIM5α is also required for HIV-1 restriction due to the requirement of TRIM5α to oligomerize [80]. This example illustrates how the N-terminal
RBCC and C-terminal PRY-SPRY domains work together and build a functional TRIM oligomer that recognizes and binds to the oligomeric HIV-1 capsid. Moreover, the RBCC domain structure has evolved as a unique block early in metazoan evolution often encoded by a single exon, suggesting that the ability of TRIMs to oligomerize was critical for human evolution [64]. The oligomeric properties of TRIMs and viral proteins may be the key to their interactions and functions.
The TRIM proteins that do not contain a PRY-SPRY domain contain C-termini with varied domains that make up the smaller subgroups of the TRIM family. The TRIM subfamily of 12
Transcription Intermediary Factors (TIFs), TRIM24 (TIF1alpha), TRIM28 (TIF1beta), TRIM33
(TIF1gamma), and TRIM66 (TIF1delta, lacking RING domain) comprise a C-terminal chromatin binding unit consisting of a Plant Homeodomain (PHD) and bromo-domain (Group VI). The tandem PHD and bromo-domains within TRIM24 and TRIM33 act as a single functional unit allowing recognition of a non-canonical histone signature combining unmethylated lysine 4 with acetylated lysine 23 or 18 of histone H3 [70, 81]. In contrast, TRIM28 possesses an atypical PHD domain that was shown to catalyze sumoylation of the adjacent bromo-domain altering the capacity of TRIM28 to function as a gene silencer [82]. TRIM24 and TRIM33 form a complex with and without TRIM28 revealed by their co-immunoprecipitation in HeLa cells, however the consequences of these interactions have not been defined [83]. It is likely that the interactions among the TIF subfamily of TRIMs alter and fine-tune their functions in transcription regulation.
Furthermore, these TRIM proteins also interact with TRIM proteins outside of the TIF subfamily suggesting an array of potential complexes to adjust their respective functions [74].
An additional C-terminal domain found in TRIM proteins is the NHL repeats that fold into a six-bladed β-propeller reminiscent of the β-propeller formed by WD40 repeats [84, 85].
Intriguingly, the NHL domain is not present in predicted Escherichia coli proteins, however, NHL- domain containing proteins have all been found in prokaryotes that are human pathogens suggesting a role of the NHL domain in human immunity [84]. Within the TRIM family, TRIM2,
TRIM3, TRIM32 (HT2A) and TRIM71 contain NHL domains at their C-termini (Group VII).
TRIM32 was the first TRIM-NHL protein to be discovered by its binding to the HIV-1 Tat transcription factor [86]. The NHL domain was found to be required for TRIM32 to interact with
Tat suggesting that the function of the NHL domain is for protein-protein interactions.
Furthermore, an aspartate to asparagine mutation in the NHL domain of TRIM32 appears to be the causal mutation in Limb-girdle muscular dystrophy type 2H patients illustrating the importance of the NHL 13
Figure 1.0 Human TRIM proteins
Classification of human TRIM proteins based on the nature of their C-terminal domains. The TRIM protein family is composed of 11 sub-families, from C-I to C-XI, whereas some TRIM proteins remain unclassified (UC), since they do not have a RING finger domain. NHL, NHL repeats; COS, COS box motif; FN3, fibronectin type III motif; PHD, plant homeodomain; BROMO, bromodomain; MATH, meprin and TRAF homology domain; TM, transmembrane domain; AR, acid-rich region.
14
domain to TRIM32 function and suggesting a function for TRIM32 in muscle maintenance [87]. In addition TRIM32 has been shown to enhance microRNA processing by interacting with AGO1 via the NHL domain [88]. TRIM-NHL protein NHL-2 in C. elegans and TRIM-NHL proteins Brat and
Mei-P26 in Drosophila melanogaster have also been shown to interact with AGO1 homologues and also mediate microRNA processing [89, 90]. Moreover, TRIM-NHL proteins are involved in mRNA repression and protein degradation regulating cell proliferation, differentiation and apoptosis in various tissues including muscles and neurons [68, 91, 92]. On top of the RBCC and
NHL protein-protein interaction motifs, TRIM2, TRIM3 and TRIM71 also have Filamin repeats preceding the NHL domain which is yet another dimerization motif [93]. The many intra- and extra-molecular interacting domains of TRIM-NHL proteins illustrates the necessity of oligomerization among the TRIMs for their diverse functions.
Finally, the COS box, a domain necessary for microtubule binding, is found within ten
TRIMs (Group I, II and III). MID1 (TRIM18) and MID2 (TRIM1) are the most well studied TRIMs containing the COS box domain and have been found to be required for neural tube closure in
Xenopus through the regulation of microtubule organization at the dorsal midline [94].
Furthermore, mutations in MID1 are responsible for the X-linked form of Optiz syndrome, a neurological disorder, where MID1 promotes neuronal axon development by regulating the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac) [95]. Within COS box containing TRIMs a Fibronectin type III motif (FN3) or a region rich in acidic amino acids follows the COX box. The fibronectin type III repeat region is composed of varied tandem repeats of
which contain potential binding sites for DNA, heparin and the cell surface [96]. The superfamily
of sequences believed to contain FN3 repeats represents 45 different families, the majority of
which are involved in cell surface binding in some manner, or are receptor protein tyrosine
kinases, or cytokine receptors. The acidic region following the COS box is reminiscent of TRIM
like protein, RNF41 (NRDP1) that targets epidermal growth factor receptor family member ErbB3
(HER3) for degradation via its RING domain [97]. The acidic region of RNF41 is required to
interact with ErbB3 and modulates cell proliferation and differentiation [98]. RNF41 has also been 15
shown to be capable of interacting directly with TRIM32 and TRIM8, neither of which has a COS box [99]. However, TRIM32 interacts with COS box containing MID2 and TRIM8 with COS box containing TRIM46 [74]. Taken together, the many protein-protein interaction motifs contained within the family of TRIM proteins highly suggests an extremely complex and well controlled network of proteins giving rise to a vast number of complexes to precisely regulate countless cellular processes using homo- and hetero-oligomerization coupled with multiple additional protein-protein interaction domains.
TRIMs in innate immunity
The innate immune response is the first line of defense against incoming pathogens and is crucial for controlling infection. Components of the innate immune response are responsible for both recognizing incoming viruses and limiting their replication and spread. Critical innate immune responses against viruses include constitutively expressed proteins with intrinsic anti- viral properties and the inducible type I interferon (IFN-I) system. TRIM proteins are part of both the intrinsic and innate immune response where they can directly interfere with viral replication and are induced by Interferons. Intriguingly, the number of TRIMs has rapidly expanded very recently in evolution of higher eukaryotes [64, 100]. The evolutionary time frame of this expansion coincided with the emergence of traits specific for the adaptive immune system, suggesting that TRIM proteins may have evolved as an integral part of the machinery to regulate the increasingly complex immune system and fine tune cross-talk between innate and adaptive immune branches. For comparison, while humans have over 70 TRIM genes, fruit flies have only seven.
Interferons (IFNs) are the main mediators of innate immunity against viral infection, by
up-regulating the expression of many antiviral effectors within cells. Three classes of IFN have
been identified, designated types I to III, and classified on the basis of the receptor complex they signal through, and their biological activities. Type I IFNs are a vast group of cytokines produced by most cells upon viral infection and trigger a signaling cascade that leads to the induction of many genes that control virus replication and spreading. Type II IFN is produced exclusively by 16
activated T lymphocytes and natural killer cells. In a systematic analysis of TRIM gene expression in human primary lymphocytes and macrophages, IFNs I and II were found to regulate gene expression of 27 TRIM genes [62]. In a separate study, a subgroup of TRIM genes were activated in mouse cells upon influenza virus infection in an IFN I dependent manner [101].
Furthermore, induction of TRIM22 by IFN I was shown to significantly inhibit HIV replication by interfering with the HIV Gag coat polyprotein [102]. The regulation of TRIM proteins by IFNs is highly suggestive that the family functions to inhibit general virus infection. Thus far, the majority of anti-viral activities of TRIM proteins have been reported to inhibit retroviruses and have not been researched for a role in DNA virus replication.
The discovery that TRIM5α from Old World monkeys is able to restrict HIV-1 replication
sparked the investigation into the anti-retroviral activities of TRIM proteins. As discussed earlier,
TRIM5α from Old World monkeys interacts with the incoming HIV-1 viral capsid polyprotein Gag
and promotes its premature uncoating and disassembly [78]. Systematic testing of the anti-
retroviral activities of TRIM proteins revealed that ~20 TRIM proteins demonstrate anti-retroviral
activity at various stages of the virus life cycle including at early and late stages of the retroviral
life cycle [61]. Intriguingly, the antiviral activity of TRIM11 was critically dependent on a functional
E3 ligase domain, implying the involvement of the ubiquitin-dependent degradation pathway. In
contrast, the E3 mutant of TRIM15 largely retains its inhibitory activity and was shown to interact
with the MLV Gag precursor protein disrupting viral capsid formation [61]. Taken together with
the knowledge that TRIM proteins form homo- and hetero- oligomers through their many protein-
protein interaction domains, it is likely that TRIM anti-viral functions occur via detection of
oligomeric virus proteins such as the polyprotein Gag, and that they somehow work together to
extinguish viral replication.
17
TRIMs in cancer
A subset of the TRIM family of proteins is deregulated in carcinomas and leukemias.
PML (TRIM19) and TRIM24 are involved in chromosomal translocations resulting in fusion proteins with RARα and the fibroblast growth factor receptor 1 (FGFR1), respectively [103].
These oncogenic fusion proteins result in the progression of acute promyelocytic leukemia (by
PML-RARα) or 8p11 myeloproliferative syndrome (EMS) (by TRIM24-FGFR1). Furthermore, it
has been reported that BRAF and RET can be fused to TRIM24 by translocation in solid tumors
[104, 105]. TRIM24 is a regulator of several nuclear receptor proteins including RARα, thyroid,
vitamin D3 and oestrogen receptors. Furthermore, TRIM24 is maintained at high levels in
myeloid progenitor cells when granulocyte differentiation is induced by RA, indicating that
TRIM24 expression is required for myeloid differentiation [106]. TRIM24 also acts as a potent
liver-specific tumour suppressor by attenuating RARα mediated transcription and that TRIM24
knock-out mice show a high incidence of hepatic tumours [107]. Moreover, hepatic cell
carcinoma formation by loss of TRIM24, is strongly enhanced by further loss of TRIM33, which is
a major interacting parter of TRIM24 [83]. TRIM33 is also a tumor suppressor in chronic
myelomonocytic leukemia (CMML) where TRIM33 is undetectable in leukemic cells of 35% of
CMML patients due to epigenetic silencing at the TRIM33 promoter [108]. Loss of TRIM33 also
cooperates with K-ras activation to induce cystic tumors and adenocarinomas of the pancreas in
mice [109]. However, the mechanism underlying the tumor suppression functions of TRIM33
remains unclear.
TRIM27, also known as RFP is another TRIM protein involved in a chromosomal
translocation leading to tumorigenesis. The N-terminus of TRIM27 and the catalytic domain of
the receptor tyrosine kinase, RET, are fused leading to an increase in catalytic activity by the
hybrid TRIM27-RET protein. The fusion protein is thought to result in ligand-independent kinase
activation and subsequent cell proliferation and tumorigenesis [110, 111]. However, TRIM27 can
also act as a tumor suppressor by inducing apoptosis through a mechanism involving activation
of Jun N-terminal kinase and p38 kinase [112]. Furthermore, it was shown that the N-terminal 18
TRIM domains of TRIM27 were sufficient to induce apoptosis[112] suggesting that TRIM-TRIM interactions may be involved in the mechanism.
TRIM32, a member of the NHL family of TRIMs, and regulates neural differentiation where the protein becomes polarized in mitosis and concentrated in one of the daughter cells
[88]. TRIM32 also ubiquitylates and degrades MYC, suggesting that this is one mechanism through which TRIM32 controls neural differentiation. Furthermore, it has been shown that the over-expression of TRIM32 in mouse neuroblastoma cells and embryonal carconima cells promotes the stability of RARα, resulting in the enhancement of neural differentiation [113]. The
TRIM32 homologue in Drosophila melanogaster is a brain tumor suppressor named brat that inhibits cell growth of neurons and causes tumor formation when mutated in flies [114]. Taken together, these data suggest a critical role of TRIM32 in human brain cancers, although its precise mechanisms of tumor suppression are unknown.
Finally, several TRIM proteins have been shown to directly regulate the tumor suppressor p53. TRIM24 binds directly to p53 mediating its ubiquitylation and degradation, while TRIM28 interacts with MDM2 and cooperates to promote p53 degradation [115, 116]. TRIM29, which does not have a RING domain has been reported to bind and antagonize p53 function [117, 118], and also regulates p53 acetylation via inhibition of Tip60, a cellular acetyltransferase protein. In addition, TRIM29 functions as a tumor suppressor in breast cancers [119] and conversely functions as an oncogene in pancreatic cancers through activation of the Wnt pathway [120]. p53 can also regulate the transcription of some TRIM proteins such as TRIM22 [121] and TRIM32
(unpublished data). TRIM22 is also an interferon regulated gene which suggests an overlap of p53 and interferon regulation of TRIM genes, although this has yet to be researched.
As a family, TRIM proteins regulate numerous diverse pathways to promote cell growth,
cell death, cell differentiation and cellular responses to invading pathogens. In Chapter 3, I
analyze the interactome of TRIM proteins and their first order interactors to identify the top Gene
Ontology Biological Processes of the TRIM family. The TRIMs undoubtedly work together to
maintain cellular protection against tumor mutations and pathogens. In Chapter 3 I show that 19
adenovirus E4-ORF3, HSV-1 ICP0, and Cytomegalovirus IE2, virus proteins known to target
PML, interact with a subset of the TRIM family using a cell based immunofluorescence assay.
Furthermore, I illustrate that these three disparate virus proteins do not mislocalize TRIM proteins using a common domain within TRIMs. Super-resolution microscopy, shown in Chapter 3, illustrates the unique subcellular localizations of E4-ORF3 and ICP0 with multiple TRIM proteins simultaneously and suggests that TRIM-TRIM inter-family interactions contribute to the observed
TRIM-viral protein interactions. Taken together, the data shown in Chapter 3 provide a unifying theme, that disparate viruses target PML as part of a much broader strategy in targeting TRIM proteins for pathological replication. Furthermore, these data will change a longstanding paradigm that PML is the central target of viruses and tumor mutations, to now include convergence on the family of TRIM proteins.
Final Introductory Thoughts
Virology, the study of viruses, is fundamentally interconnected with the study of the host cell. The discovery of virus protein interactions with cellular proteins has, and will continue to reveal critical proteins that regulate cell growth and survival. The hard work of scientists has delivered the human race with new types of therapies using viruses as vectors for gene delivery or as potent killing machines. The work in this dissertation will advance the knowledge of DNA viruses to ultimately be used to treat patients and possibly to engineer drugs to target viral pathogens and protect against pathogenic infection.
Chapter 1. Heterochromatin silencing of p53 target genes by a small viral protein
Abstract
The transcription factor p53 guards against tumor and virus replication and is inactivated in almost all cancers. p53 activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and DNA damage. During adenovirus replication, the degradation of p53 by E1B-55k is considered essential for p53 inactivation, and is the basis for p53 selective viral cancer therapies. Here we reveal a dominant epigenetic mechanism that silences p53 activated transcription, irrespective of p53 phosphorylation and stabilization. We show that another adenoviral protein, E4-ORF3, inactivates p53 independently of E1B-55k by forming a novel nuclear structure that induces de novo H3K9me3 heterochromatin formation at p53 target promoters, preventing p53 DNA-binding. This suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. These findings are important for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumor suppressor loci. Our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53 selective oncolytic viral therapies.
Introduction/Results
Tumor mutations and DNA virus proteins functionally converge in inactivating p53 [122], which was initially discovered as the cellular target of SV40 Large T [10, 11]. However, despite 30 years of research, the critical factors that determine p53 activated transcription are still not fully understood [123, 124]. p53 is expressed constitutively in normal cells where its activity is limited by highly regulated p53 protein degradation [125]. p53 activation is triggered in response to oncogenes and DNA damage, which stabilize p53 [126-128]. This has led to the general belief that the induction of p53 levels and phosphorylation [129] is synonymous with p53 activated transcription of target promoters in cellular chromatin. As such, the induction of p53 levels is a standard read-out for p53 activation and rationale for several cancer therapies, including
20 21
irradiation and genotoxic drugs [130], MDM2 antagonists [20], and the E1B-55k deleted oncolytic adenoviral therapy, ONYX-015 [131].
The adenoviral protein, E1B-55k, binds to the p53 transactivation domain and is sufficient to inactivate p53 in cellular transformation [126, 132]. In adenovirus infection, E1B-55k also interacts with another viral protein, E4-ORF6, which recruits a cellular ubiquitin ligase complex that targets p53 for proteosomal degradation [32, 133]. The degradation of p53 by E1B-55k is thought to be the critical event that inactivates p53 for virus replication [134]. As such, the induction of high p53 levels in response to an E1B-55k deleted virus[135], dl1520/ONYX-015, was expected to prevent viral replication in normal cells but not p53 mutant tumor cells [131]. On this basis, ONYX-015 [131] was tested in cancer patients as a p53 tumor selective oncolytic viral therapy [136, 137] and is now approved in several countries (now known as Oncorine). However, the loss of E1B-55k functions in viral RNA export, rather than p53 inactivation, is the major determinant of ∆E1B-55k tumor selectivity [138, 139]. Contrary to expectations, although p53 accumulates to high levels in the nucleus of ∆E1B-55k (∆55k) infected human primary small
airway epithelial cells (SAECs), the physiological target cells for adenovirus infection, p53
transcriptional targets, such as, p21, MDM2, Cyclin G, 14-3-3σ, PERP, PIG3 and GADD45 are
not induced (Figure 1.1a and ref [138]). The failure of p53 stabilization to activate p53
transcriptional targets is not a tissue-specific effect and occurs in different primary cell types and
tumor cell lines infected with ∆E1B-55k (Supplementary Figure 1.1-3), including p53 wild-type
U2OS tumor cells where p53 targets are suppressed to a similar extent as that in cell-lines with
p53 mutations. This reveals a fundamental gap in our understanding of not only adenovirus
biology but also p53 activation.
p53 stabilization without activity
Cellular and viral oncogenes, such as Ras and Adenovirus E1A, trigger p53 activation by
inducing the expression of ARF [128], which inhibits MDM2 mediated p53 degradation. ARF is
lost in 58% of cancers[128], which had previously been invoked as the critical factor that prevents
p53 activation in ∆E1B-55k infected tumor cells [140]. Using a U2OS stable cell-line (p53 wild- 22
type, ARF negative) in which ARF expression is induced by IPTG [141], we show that ARF stabilizes p53 and activates p21 transcription in mock infection. Nevertheless, despite the induction of ARF and basal p53 activity, p21 levels are still repressed to similar levels in both wild-type (wt) and ∆E1B-55k infected cells (Figure 1.1b and Supplementary Figure 1.4).
Furthermore, endogenous ARF induction also fails to activate p53 targets in ∆E1B-55k infected
SAECs (Figure 1.1a). Thus, p53 is inactivated, irrespective of E1B-55k and ARF expression in adenovirus infected cells.
DNA damage signals also play a critical role in activating p53, triggering p53 phosphorylation and protein stabilization [127, 142]. In clinical trials, ∆E1B-55k (ONYX-015), was used in combination with genotoxic chemotherapies, such as 5-fluorouracil (5-FU) [137, 143]. We reasoned that the induction of p53 levels alone may not be sufficient to activate p53 in ∆E1B-55k infected cells, and that DNA damage is also required. However, 5-FU fails to activate p53 in
∆E1B-55k infected U2OS cells (Figure 1.1c). The DNA damage checkpoint is deregulated in many tumor cells. Therefore, we also analyzed ∆E1B-55k infected SAECs and show that p53 transcriptional targets cannot be activated by γ irradiation (Figure 1.1d and Supplementary Figure
1.5), UV irradiation (Supplementary Figure 1.6) or doxorubicin (dox, Figure 1.1f).
The activation of p53 in response to DNA damage is mediated via kinases, such as ATM,
ATR, DNA-PKcs, CHK1 and CHK2, which phosphorylate p53 [127] at key residues, stabilizing
p53 and potentiating p53 DNA binding [142]. A possible explanation for the failure of DNA
damage to activate high p53 levels in ∆E1B-55k infected cells is that p53 phosphorylation is being
inhibited in viral infection. However, even without the introduction of exogenous genotoxic stress,
p53 is already highly phosphorylated at multiple sites targeted by DNA damage activated kinases
in ∆E1B-55k infected SAECs (Figure 1.1e). Thus, although oncogenes and DNA damage trigger
p53 stabilization and phosphorylation in ∆E1B-55k infected cells, p53 fails to activate the
transcription of downstream effectors.
We next examined if in the absence of E1B-55k, MDM2 binds and inactivates p53 in
adenovirus infected cells. Nutlin is a small molecule antagonist of MDM2 that inhibits MDM2-p53 23
binding [20]. In contrast to mock, nutlin fails to stabilize p53 further or induce p21 in ∆E1B-55k
infected SAECs (Figure 1.1f). The histone deacetylase (HDAC) inhibitor, trichostatin A (TSA),
induces the expression of p21 independently of p53 stabilization or phosphorylation (Figure 1.1f).
However, in ∆E1B-55k infected cells, TSA fails to induce the transcription of p21. We conclude
that p53 transcriptional targets are dominantly suppressed in adenovirus infected cells,
irrespective of E1B-55k, and cannot be activated in response to radiation, genotoxic drugs, ARF,
MDM2 antagonists or HDAC inhibitors.
E4-ORF3 inactivates p53 independently of E1B-55k
Our data strongly suggest that there is a previously undiscovered adenoviral protein that
inactivates p53 independently of E1B-55k and p53 degradation. To test this, we used a genetic
approach and screened for p53 activation in primary cells infected with adenoviruses that have
compound mutations in E1B-55k and other early viral genes (detailed explanation of viruses in
Supplementary Figure 1.7). In addition to deleting E1B-55k, the loss of either E1A-13s or E4-
ORF3 is required to activate p53 in infected cells (Figure 1.2a). Our discovery of two additional
viral proteins that inactivate p53 is surprising, especially since E1A is a potent oncogene that
triggers p53 activation in cellular transformation studies [126, 132]. In adenovirus infection, the
alternative 13s splice form of E1A is required for the transactivation of other viral genes [134],
including E4-ORF3 (Figure 1.2a). Therefore, we hypothesized that E1A-13s inactivates p53 by
inducing the expression of E4-ORF3 in viral infection. Consistent with this, we show that in
contrast to GFP, the ectopic expression of E4-ORF3 rescues p53 inactivation in both ∆E1B-
55k/∆E4-ORF3 and ∆E1B-55k/∆E1A-13s infected cells (Figure 1.2b). The slight reduction of p21
by Ad-GFP in ∆E1B-55k/∆E4-ORF3 co-infected cells is due to the partial activation of E4-ORF3
transcription (in trans) by E1A-13s, which does not occur in ∆E1B-55k/∆E1A-13s co-infection
(Supplementary Figure 1.8). Hence, E1A-13s induces the expression of E4-ORF3 which then
inactivates p53 via an E1B-55k independent mechanism. Moreover, the expression of E4-ORF3
in either SAECs or U2OS cells is sufficient to inhibit p53 activation in response to nutlin and
doxorubicin, respectively (Supplementary Figure 1.9). These data reveal E4-ORF3 as a novel 24
adenoviral protein that inactivates the p53 tumor suppressor pathway.
The proposed p53 tumor selectivity of the ∆E1B-55k oncolytic viral therapy, ONYX-015
(Oncorine), is based on p53 stabilization being the critical event that determines p53 activated transcription of downstream effectors. Although there is some basal p53 activity in ∆E1B-55k infected cells compared to wild-type virus, the additional deletion of E4-ORF3 is necessary for the consequent induction of p53 levels to activate the transcription of downstream targets over the course of infection, including cell cycle arrest, DNA repair and apoptosis genes (Figure 1.2c-d). In contrast to p53 transcriptional targets, the mRNA levels of p53 and the housekeeping gene,
GUSB, are not impacted by E4-ORF3 (Supplementary Figure 1.10). p53 stabilization is required to activate p53 transcriptional targets, and does not occur in ∆E4-ORF3 infection where p53 is degraded by E1B-55k/E4-ORF6. Furthermore, using a p53 inducible stable cell-line (H1299-D1,
Supplementary Figure 1.11), we show that the induction of p21 and MDM2 in ∆E1B-55k/∆E4-
ORF3 infection is p53-dependent. These experiments also demonstrate that E4-ORF3 does not
inactivate p53 by inducing alternative p53 splice forms [144], since H1299-D1 cells express a p53
cDNA. Thus, the deletion of both E1B-55k and E4-ORF3 is necessary to activate p53 in
adenovirus infection. We conclude that E4-ORF3 has a critical and novel role in inactivating p53
independently of E1B-55k and p53 degradation.
E4-ORF3 prevents p53-DNA binding at chromatin
DNA tumor virus proteins, such as E1B-55k, SV40 LT and HPV E6 inactivate p53 via
direct high affinity protein-protein interactions [122]. However, contrary to this established
paradigm, E4-ORF3 does not co-localize with p53 (Supplementary Figure 1.12) or co-
immunoprecipitate with p53 (data not shown). This suggests that E4-ORF3 inactivates p53 via a
non-canonical mechanism.
25
Figure 1.1 p53 is induced and phosphorylated in ∆E1B-55k infection but p53 activity is dominantly suppressed a. SAECs were infected and protein lysates analyzed by immunoblotting. b. U2OS cells with inducible ARF were infected and analyzed by immunoblotting. c. U2OS cells were infected and treated 24 h.p.i. with vehicle control (-) or 5-FU. Protein lysates at 0 hours (hrs) and 36 h.p.i. were analyzed by immunoblotting. d. RT-QPCR of p53 transcriptional targets in infected SAECs (36 h.p.i.) plus/minus 10 Gy γ-irradiation (IR). e. Immunoblot of p53 protein phosphorylation in infected or doxorubicin (dox) treated SAECs (36 h.p.i.). f. SAECs were infected and treated with either control (-), dox, nutlin, or TSA at 24 h.p.i. Protein lysates (36 h.p.i.) were analyzed by immunoblotting. 26
The induction of p53 levels and phosphorylation induces p53 conformational changes that drive sequence-specific DNA binding and the recruitment of transcription co-factors [145]. A p53
DNA binding domain that is competent to bind to DNA can be distinguished by immunoprecipitation with PAb 1620 versus PAb 240 [146]. p53 is immunoprecipitated selectively by PAb 1620 in both ∆E1B-55k and ∆E1B-55k/∆E4-ORF3 infected cells (Supplementary Fig13), demonstrating that the p53 DNA binding domain is in a protein conformation that should be capable of binding to DNA [146] in both cases. To functionally determine if E4-ORF3 prevents p53 DNA binding, we transfected U2OS cells with a p53 luciferase reporter plasmid (p53-luc), where p53 binding to consensus DNA sequences activates luciferase transcription [147]. A control pGL3-luciferase reporter (non-p53 promoter) is activated to similar levels in all viral infections (Supplementary Figure 1.14). In wild-type virus infected cells, p53 activated transcription of luciferase is inhibited after 24 hours (Figure 1.3a), which is expected due to p53 degradation (Supplementary Figure 1.3b). In contrast, p53-luciferase is activated in both ∆E1B-
55k and ∆E1B-55k/∆E4-ORF3 infection (Figure 1.3a). The induction of luciferase activity requires p53 DNA binding, since a promoter with a mutated p53 response element (p53-mutant) abolishes luciferase activity. These experiments demonstrate that E4-ORF3 does not compete with p53 for binding to consensus DNA target sequences or prevent p53 transcriptional activation of promoters in ectopic reporter plasmids.
The ability of E4-ORF3 to prevent p53 activated transcription of endogenous targets but
not ectopic p53-luciferase plasmids is at first difficult to reconcile. Plasmid DNA is not subject to
the same architectural and packing constraints as DNA in cellular chromatin. Therefore, we
performed p53 chromatin immunoprecipitations (ChIPs) to determine if E4-ORF3 specifically
prevents p53 DNA binding in the context of cellular chromatin. p53 binding to target sites in the
p21 (5’ and 3’ site) and MDM2 promoters [145] is induced upon doxorubicin treatment and ∆E1B-
55k/∆E4-ORF3 infection, where it activates the transcription of p21 and MDM2 RNAs (Figure 27
Figure 1.2 E4-ORF3 inactivates p53 independently of E1B-55k and p53 degradation
a. SAECs were infected with the indicated viruses (see Supplementary Figure 1.7) and protein lysates (36 h.p.i.) analyzed by immunoblotting. b. SAECs were co-infected as indicated with either a GFP control virus (Ad-GFP, +) or a virus expressing E4-ORF3 (Ad-ORF3, +). Protein lysates at 36 h.p.i. were analyzed by immunoblotting. c. Protein lysates from infected SAECs were analyzed by immunoblotting at 0, 24, 36 and 48 h.p.i. d. RT-QPCR of p53 transcriptional targets in infected SAECs at 36 h.p.i.
28
1.3b-c). In contrast, although p53 is induced to similar levels, E4-ORF3 prevents p53 DNA binding to the p21 and MDM2 promoters in ∆E1B-55k infected cells (Figure 1.3b-c and
Supplementary Figure 1.15). Thus, E4-ORF3 inactivates p53 by preventing p53 binding to DNA
target sites specifically in the context of cellular chromatin.
Repressive histone methylation silences p53 targets
We reasoned that p53 DNA binding depends not only on the protein conformation of p53 but also
the accessibility of target promoters in the cellular genome. We hypothesized that E4-ORF3 could
inactivate p53 by inducing heterochromatin at endogenous target promoters, preventing the
access of p53 to DNA. Heterochromatin compaction is specified by the loss of histone acetylation
and induction of repressive histone methylation [148]. TSA fails to induce p21 in ∆E1B-55k
infected SAECs (Figure 1.2d) suggesting that E4-ORF3 inactivates p53 targets via a mechanism
that is dominant to the inhibition of histone deacetylation. In cancer, the aberrant epigenetic
silencing of tumor suppressor genes, such as p16INK4a, is initiated by the methylation of histone
H3 at lysine 9 (H3K9) [149]. p53 localization is indistinguishable in ∆E1B-55k and ∆E1B-55k/∆E4-
ORF3 infected cells (Figure 1.3d). However, in ∆E1B-55k infected cells, where p53 is inactive,
dense regions of H3K9me3 repressive heterochromatin are induced at the periphery of the
nucleus (Figure 1.3d and Supplementary Figure 1.16). Of the four known methyltransferases that
catalyze H3K9 trimethylation, we show that SUV39H1 and SUV39H2 (which share 59%
sequence identity and have redundant functions) [150, 151], but not SETDB1 [152] or G9a [153],
are specifically associated with the formation of de novo H3K9me3 heterochromatin domains in
∆E1B-55k infected nuclei (Figure 1.3e). The formation of these domains requires E4-ORF3 and
does not occur in either mock or ∆E1B-55k/∆E4-ORF3 infected cells (Supplementary Figure 1.17-
20).
29
Figure 1.3 E4-ORF3 induces heterochromatin formation and prevents p53-DNA binding at endogenous promoters a. U2OS cells were transfected with p53-luc (solid line) or p53-mutant (dashed line) luciferase plasmids and infected with mock, wt or ∆55k viruses. Luminescence is plotted against time. b and c. U2OS cells were infected or treated with doxorubicin. p53 induction was analyzed by immunoblotting and p53 transcriptional targets quantified by RT-QPCR (36 h.p.i.). c. p53 ChIPs were analyzed by semi-quantitative PCR for p21 and MDM2 promoter sequences. d. p53 (green) and H3K9me3 (red) immunofluorescence of infected U2OS cells (36 h.p.i.). e. Co- localization of SUV39H1, SUV39H2, SETDB1 and G9a (green) with H3K9me3 (red) in ∆55k infected U2OS cells (36 h.p.i.).
30
These data demonstrate that E4-ORF3 induces novel H3K9me3 heterochromatin, which could deny p53 access to endogenous target promoters. To test this, we performed p53 and
H3K9me3 ChIPs. The induction of repressive heterochromatin by E4-ORF3 is not associated with a global upregulation of either total histone H3 or H3K9me3, which are at similar levels in mock,
∆E1B-55k and ∆E1B-55k/∆E4-ORF3 lysates (Figure 1.4a). In ∆E1B-55k/∆E4-ORF3 infected cells,
p53 binding is induced at p21 and MDM2 promoter sites (consistent with Figure 1.3c) while
H3K9me3 is at a similar level to an IgG negative control (Figure 1.4a and Supplementary Figure
1.21). In contrast, in ∆E1B-55k infected cells, H3K9me3 is enriched at the p21 and MDM2
promoters where p53 binding is prevented. H3K9me3 is also induced at the -5kb region of the
p21 promoter and is not restricted to p53 binding sites (Supplementary Figure 1.21). Thus, in
cells expressing E4-ORF3, there is an inverse correlation between p53 and H3K9me3 at p53
regulated promoters. The same conclusions were reached for additional p53 targets, including
GADD45A, FAS, PUMA and PIG3 (Supplementary Figure 1.21-23). In contrast, at non-p53
regulated promoters, such as ACTIN and POLR2, H3K9me3 is not induced in ∆E1B-55k infected
cells relative to mock (Supplementary Figure 1.21 and 23). Basal H3K9me3 is decreased at these
promoters in ∆E1B-55k/∆E4-ORF3 infected cells, suggesting that E4-ORF3 may also restrain
global demethylase activity. We conclude that E4-ORF3 inactivates p53 by inducing de novo
H3K9me3 heterochromatin silencing at p53 target promoters. With access denied, p53 is
powerless to activate the transcription of downstream effectors.
The induction of heterochromatin formation is still relatively poorly understood. Thus, a
major question is how is E4-ORF3 directly involved in inducing repressive H3K9me3
heterochromatin at p53 target promoters? E4-ORF3 does not co-localize with p53 and forms a
distinctive web-like structure in the nucleus (Supplementary Figure 1.12). We show that E4-ORF3
demarcates the formation of de novo H3K9me3 heterochromatin domains in both ∆E1B-55k
infected tumor and primary cells. Orthogonal slices through the nucleus reveal that E4-ORF3 is,
for the most part, adjacent to H3K9me3, suggesting it acts as a novel platform that catalyses
heterochromatin formation through transient or long-range interactions (Figure 1.4b-c, and 31
Supplementary Figure 1.24-25). Using high resolution confocal microscopy, we show that E4-
ORF3 forms a continuous scaffold that organizes and specifies de novo heterochromatin assembly as it weaves through the nucleus (Figure 1.4d). These data demonstrate a direct role for E4-ORF3 in orchestrating H3K9me3 heterochromatin silencing at p53 target promoters.
Furthermore, they reveal an extraordinary nuclear scaffold that either builds on existing architectural features that organize cellular DNA or is a novel viral construction that targets heterochromatin assembly at p53 target promoters.
To determine if H3K9 trimethylation via SUV39H1 and SUV39H2 are necessary for E4-
ORF3 mediated silencing of p53 activity, we performed RNAi knockdown experiments in U2OS cells. Using previously published and validated RNAi sequences for SUV39H1 [154] and
SUV39H2 [155], we were able to achive knockdown of SUV39H1, as evidenced by Western blotting oflysates from transfected cells (Figure 1.5c), and SUV39H2 at the RNA level analyzed by Q-PCR (Figure1.5a) (our SUV39H2 antibodies only recognize the native folded protein).
Given that SUV39H1 and SUV39H2 have redundant functions, we transfected cells with both
SUV39H1 and SUV39H2 siRNA. We show that, the knockdown of SUV39H1, SUV39H2, or both together did not result in an appreciable decrease in the levels of H3K9me3 in transiently transfected cells (Figure 1.5c). As such, it also did not prevent p53 inactivation in ∆E1B-55k infection (Figure 1.5b). This result is not surprising as there is not a global induction of H3K9me3 associated with E4-ORF3 induced silencing of p53 target genes, and it is likely that low levels of
SUV39H1 and SUV39H2 are sufficient to catalyze H3K9 trimethylation. Taken together, these data suggest that SUV39H1 and SUV39H2 induced H3K9me3 may not be required to silence p53 target genes, and instead may be a later step to maintain the heterochromatin state.
32
Figure 1.4 E4-ORF3 forms a nuclear scaffold that specifies heterochromatin assembly and H3K9 trimethylation at p53 target promoters. a. Protein lysates from infected U2OS cells (36 h.p.i.) were analyzed by immunoblotting. H3K9me3 and p53 ChIPs were quantified by RT-QPCR, normalized relative to input DNA and plotted as fold change relative to mock. b and c. Confocal images of ∆55k infected U2OS and SAECs at 36 h.p.i. H3K9me3 (green), E4-ORF3 (red) and DNA (white). Central z-plane of a 3D-stack (right panel) with dashed lines representing orthogonal cuts that are depicted as flat projections (bottom and right side). d. Magnified high resolution confocal slice of ∆55k infected SAECs. 33
Selective silencing of the p53 transcription program
These data beg the question as to the specificity of E4-ORF3 in silencing p53 targets. To determine the global consequences on cellular transcription, we performed genome-wide expression analyses on infected SAECs (Supplementary Figure 1.26 and 27). These studies demonstrate that E4-ORF3 is an exclusive player in the global transcriptional changes induced upon viral infection. There are 1,730 overlapping genes that are similarly up or downregulated by a log fold change (FC) greater than two in both ∆E1B-55k and ∆E1B-55k/∆E4-ORF3 versus
mock, which reflect a common transcriptional program (Figure 1.6a). These global changes in
gene expression are associated with the cell cycle and E2F activation (Supplementary Table I
and II). This is consistent with E1A mediated inactivation of RB [9] and recruitment of p300 and
PCAF to induce active histone acetylation marks (H3K18acetyl) at the promoters of genes
involved in cell growth, division and DNA synthesis [156-158]. Thus, E4-ORF3 induced
heterochromatin silencing, as well as the scaffold it forms throughout the nucleus, does not affect
the global activation of cellular transcripts induced by viral infection.
To define the genes specifically targeted by E4-ORF3, we compared ∆E1B-55k/∆E4-ORF3
versus ∆E1B-55k infected cells. E4-ORF3 prevents the transcriptional activation of 265 genes by
a log fold change of two or more in ∆E1B-55k infected cells (there are 106 differentially down-
regulated genes). To determine how many of the 265 genes are likely to be regulated by p53, we
used two criteria: the presence of consensus p53 DNA binding sites in their promoters and their
induction upon treatment with the MDM2 antagonist, nutlin. A heat map of top transcripts
differentially upregulated in response to ∆E1B-55k/∆E4-ORF3 and nutlin includes well known p53
targets (MDM2, FAS, PIG3, TP53INP1, BTG2, LRDD/PIDD) associated with growth inhibition and
apoptosis, as well as novel targets (HRH1, RNASE7, JMJD1C) (Figure 1.6b and Supplementary
Figure 1.28). We show that 73% of the 265 differentially upregulated genes are induced in
response to nutlin and/or have predicted p53 binding sites while 27% do not fall into either of
these categories (Figure 1.6c and Supplementary Table III-IV). A pathway analysis of E4-ORF3
regulated transcripts indicates that in addition to the p53 pathway, there is a 34
Figure 1.5 Transient knockdown of SUV39H1 and SUV39H2 is not sufficient to decrease H3K9me3 levels and prevent E4-ORF3 induced silencing of p53 transcriptional targets.
U2OS cells were transfected with negative control siRNA, or SUV39H1 (H1) and SUV39H2 (H2) siRNA for 24 hours, and then infected with mock, ∆55k, or ∆55k/∆ORF3 viruses for 36 hours. Real-Time PCR was performed to check for knockdown of a. SUV39H1 and SUV39H2, as well as for b. p53 targets MDM2 and p21, plotted as fold change with respect to (wrt) mock cells transfected with control siRNA. c. Samples for single and double siRNA knockdown were lysed in ChIP lysis buffer and Western blotted for SUV39H1 and H3K9me3 protein levels.
35
significant overrepresentation of genes associated with immune modulation as well as tissue/vascular remodeling (Supplementary Table V). These data suggest that E4-ORF3 may target p53 promoters as part of a general anti-viral transcriptional silencing program, which is consistent with the highly defective replication of ∆E1B-55k/∆E4-ORF3 in primary cells
(Supplementary Figure 1.29).
Discussion and perspective
The conclusions of our study challenge the general assumption that p53 induction and
phosphorylation is tantamount to p53 activity, which is the premise for several cancer therapies
[20, 130, 131]. Our data reveal a novel and dominant mechanism of p53 inactivation that acts via
the targeted epigenetic silencing of p53 target promoters. We identify a viral protein, E4-ORF3,
which appears to form a novel scaffold that weaves through the nucleus, directing SUV39H1/2
H3K9me3 heterochromatin assembly at p53 target promoters to silence p53 activated
transcription in response to genotoxic and oncogenic stress (Figure 1.6d). Remarkably, this
suppressive nuclear web selectively ensnares p53 and anti-viral genes and operates in the
backdrop of global transcriptional changes that drive pathological cellular and viral replication.
There is a profound functional overlap between adenovirus early proteins and tumor
mutations [159], which has led to the identification of many of the key growth regulatory
mechanisms, including E2F and p300. Thus, a major question is if E4-ORF3 reflects or exhorts
an existing mechanism and nuclear structure that censors p53 transcriptional activity in normal
cells or tumorigenesis. Strikingly, all of the known cellular targets of E4-ORF3, PML [160], the
MRE11/RAD50/NBS1 (MRN) DNA damage/repair complex [39] and Tif1α [41]are subverted by
tumor mutations. It is intriguing to speculate that E4-ORF3 physically integrates the inhibitory
effects of several cancer pathway mutations, both known and yet to be discovered, which
together have emergent functions [123] in silencing p53 activity. Similar to the discovery of p53
with a viral protein [10, 11], E4-ORF3 provides a powerful dynamic probe with which to define
critical cellular factors that induce de novo and targeted epigenetic silencing of p53 target 36
Figure 1.6 p53 transcriptional targets are silenced selectively in the backdrop of global transcriptional changes that drive oncogenic cellular and viral replication. a. Affymetrix global gene expression analyses of infected SAECs (36 h.p.i.). Heat map of the 1,730 overlapping differentially regulated genes (log FC>2 or <-2 with a false discovery rate (FDR) of 0.05) between ∆55k/∆ORF3 and ∆55k versus mock. b. Unsupervised hierarchical clustering of 46 top differentially upregulated transcripts in both ∆55k/∆ORF3 infection and nutlin. c. Pie-chart depicting the percentage of upregulated transcripts (log FC>2 and FDR of 0.05) in ∆55k/∆ORF3 versus ∆55k that have predicted p53 transcription factor binding sites (TFBS) and/or induced by a log FC>1.5 in response to nutlin. d. Summary and model.
37
promoters in somatic human cells. This has important implications for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the dynamic mechanisms that induce aberrant epigenetic silencing of tumor suppressor gene loci. Finally, our identification of E4-ORF3 changes the fundamental definition of how p53 is inactivated in adenovirus infected cells, which is a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies.
Methods Summary
Primary cells and tumor cell-lines were cultured and infected with established conditions
[138, 139]. Protein lysates were normalized and analyzed by Western blotting [138, 139]. Real-
Time Q-PCR (RT-QPCR) was used to quantify p53 transcriptional targets [138], which were normalized relative to 18S. Error bars representing the standard deviation across triplicates.
Protein localization and chromatin remodeling was visualized by immunofluorescence [138, 139].
For luciferase assays, U2OS cells were transfected with reporter plasmids and infected after 36 hours. 100 uM D-Luciferin was added 4 hours post infection (h.p.i.) and luminescence quantified every hour. p53 and H3K9me3 ChIPs were [145, 161] analyzed using either semi-quantitative
PCR or RT-QPCR. Global gene expression of infected SAECs was performed using Affymetrix
Human Exon 1.0ST arrays and analyzed with Partek and Genomatix software.
Extended Methods
Cells, culturing conditions and viral infections.
Primary human cells from multiple donors were obtained from Cambrex/Lonza, which were cultured and infected as described previously [138, 139]. Multiplicity of infections (M.O.I.) were determined experimentally. U2OS, H1299-D1, MDA-MB-231, HCT-116, C33A and A549 cells were infected at M.O.I. 30, and SAECs, HMEC and HBEC at an M.O.I. of 10. U2OS cells with an IPTG inducible ARF [141] and were a kind gift from Dr. Gordon Peter’s laboratory. H1299-
D1 is a stable cell-line that expresses a p53 cDNA construct under the control of a ponasterone inducible promoter. To induce p53 expression, cells were treated 5 hours prior to infection with 5
μM ponasterone A (Invitrogen), which was re-added at the time of infection. 38
Viruses and viral replication assays.
Viral replication and titers were quantified by secondary infection of 293/E4/pIX cells using an ELISA assay with a rabbit anti-Adenovirus Type 5 antibody (Abcam) at 1:1000, as described previously [138, 139], [162]. Mock infection was performed with the E1 deleted non- replicating adenovirus dl312 [163]. Wild-type (wt) virus is WtD [135], ∆E1B-55k (∆55k) is dl1520/ONYX-015 [131, 135]. The ∆E1B-55k/∆E4-ORF3 (∆55k/∆ORF3) virus, dl3112, has an identical genome backbone to dl1520/ONYX-015 but has a single base pair deletion (nucleotide
7143R) that ablates E4-ORF3 expression [164]. ∆E1B-55k/∆E4-ORF2 (∆55k/∆ORF2) has been described previously [164]. ∆E4 is dl366 which has a deletion that ablates the E4 genes [165].
∆E1B-55k/∆E4-ORF6 (∆55k/∆ORF6) is dl367 which has mutations that ablate both E1B-55k and
E4-ORF6 [166]. ∆ORF3 is E4inORF3 [167]. ∆E1B-55k/∆E1A-13s (∆55k/∆13s) has an E1B-55k
gene deletion and mutation that ablates the 13s splice form of E1A [168]. ∆E1B-55k/E1A∆p300
(∆55k/E1A∆p300) has an E1B-55k deletion and E1A point mutation that abrogates E1A-p300
binding [138, 168]. Ad-CMV E1 deleted replication incompetent adenovirus vectors (Ad-CMV-
Dest, Invitrogen) were constructed to express either E4-ORF3 (Ad-ORF3) or GFP (Ad-GFP).
Plasmids, Drugs and DNA damage.
pDONR221 plasmid for SUV39H1 was purchased from the Harvard Institute of
Proteomics (Plasmid ID: HsCD00044660) and cloned into a CMV expression vector in frame with
an N-terminal myc tag. Doxorubicin (dox, Sigma) at 0.5 μg/ mL for 12 hours was used as a
positive control for p53 activation. 5-fluorouracil (5-FU, Sigma) was used at 50 μg/mL, trichostatin
A (TSA, Sigma) at 5 μM and nutlin-3 (Calbiochem) at 10 μM. γ irradiation was performed via
exposure to a cobalt-60 source.
Protein lysates and Western analysis.
Protein lysates were harvested, normalized and analyzed as described previously [138,
139]. Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, GFP,
MDM2 N20), Cell Signaling Technology (phospho-p53 serine (ser) 6, 9, 15 (16G8), 20, 33, 46,
315, 392 and threonine (thr) 81), Upstate (p21), Calbiochem (p53 PAb 1620 and 240, MDM2 39
2A10), Abcam (Actin and histone H3), Active Motif (H3K9), Ascenion (E4-ORF3 (6A11)), ARF
[138], and E1B-55k (2A6). Actin expression was used as a loading control. Primary antibodies were detected with secondary antibodies labeled with either IRDye800 (Rockland), Alexa Fluor
680 (Molecular Probes) or HRP-conjugated secondary antibodies. Fluorescent antibodies were visualized using a LI-COR-Odyssey scanner and HRP antibodies with ECL chemiluminescence followed by autoradiography.
Real-Time Quantitative PCR Analysis.
Real-Time Q-PCR (RT-QPCR) quantification of p53 transcriptional targets was performed using an ABI Prism 7900 system, as described previously [138]. 1 μg of total RNA was reverse transcribed with Applied Biosystems’ High Capacity Reverse Transcription. RT-QPCR reactions were set up using Taqman Fast mix (ABI), and run in triplicate. 10 ng of input cDNA was used for
18S analysis, and 50 ng of input cDNA was used for all other targets. All samples were normalized to 18S expression.
Immunofluorescence.
Cells were fixed in 4% paraformaldehyde and stained as described previously [138, 139].
Primary antibodies were from Santa Cruz Biotechnology (p53 DO-1 and FL393, SUV39H2 Z-23),
Cell Signaling Technology (SETDB1/ESET, G9a/EHMT2), Diagenode (H3K9me3 mouse), Active
Motif (H3K9me3 rabbit), Abcam (SUV39H2, SUV39H1), and E1A (M73), Roche (myc 9E10), and
Ascenion (E4-ORF3 (6A11)). Alexa 488, 555 and 633 (Molecular Probes) conjugated secondary antibodies were used for detection of primary antibodies. Images were acquired with a Zeiss
Axioplan2 imaging system (Supplementary Figure 1.3a, 12, 16 and 19), a Nikon A1 laser confocal system (Figure 1.3d-e, 4b-d, Supplementary Figure 1.20 and 24) and a Leica confocal SP2
(Supplementary Figure 1.17, 18 and 25). Pictures were edited in Adobe Photoshop. Background was corrected using same gray level values for all channels to remove noise, except for reducing the contrast of SETDB1 staining for the ∆55k/∆ORF3 panel in Supplementary Figure 1.20. The raw microscope images acquired with the Nikon A1 laser confocal system were filtered with a 5 kernel median filter. 40
Real-Time Luciferase Assays.
U2OS cells were reverse transfected with luciferase reporter plasmids, p53-luc, p53- mutant and pGL3 (Stratagene) using Fugene 6 (Roche). After 36 hours, cells were infected with viruses. After four hours, media was replaced by high glucose DMEM without phenol red, supplemented with 15 mM HEPES pH 7-7.4 (Gibco) and 100 uM D-Luciferin (BIOSYNTH
Chemistry & Biology). Plates were sealed with vacuum grease (Dow Corning) and custom-made glass cover slips. Luciferase activity was analyzed using the temperature-controlled Safire II plate reader with Magellan 6 software (Tecan Group, Ltd). The luminescence for each well was integrated over 4 seconds and read at 1 hour intervals for 48 hours at a temperature setting of
37oC. Data was graphed in Excel 2007 (Microsoft) by plotting the average luminescence of the
triplicates at each time point.
Chromatin Immunoprecipitation Assays.
p53 and H3K9me3 chromatin immunoprecipitations (ChIPs) were performed as
described previously [145, 161]. Cells were fixed with 1% formaldehyde for 15 minutes and
stopped with 0.125 M glycine, lysed in Szak’s modified RIPA buffer or 1% SDS lysis buffer and
sonicated to shear genomic DNA. p53 and H3K9me3 ChIPs were performed using 2 μg of p53
DO-1 monoclonal antibody and 5 uL of anti-H3K9me3 rabbit serum (Active Motif), respectively. A
mouse IgG isotype control and non-immune rabbit serum were used as controls for specificity.
Immuno-complexes were isolated using Protein G (mouse antibodies) or Protein A beads (rabbit
antibodies). Crosslinking was reversed by incubating at 65oC overnight. DNA was purified and analyzed using either semi-quantitative PCR or RT-QPCR using previously described conditions and primers [145, 169, 170]. For RT-QPCR, samples were analyzed in duplicate using a Sybr
GreenER mix (Invitrogen) and quantified on a MyiQ RT-QPCR machine (Bio-Rad). A 10-fold dilution series of input DNA was used to determine the efficiency of the PCR for each primer set.
ChIP DNA samples were normalized relative to their respective input DNAs.
41
Affymetrix expression arrays and data analysis.
Human primary quiescent SAECs were infected and harvested at 36 h.p.i. All samples were done in duplicate, and corresponding lysates were western blotted. Total RNA was isolated and purified using Trizol with the PureLink RNA Mini kit (Invitrogen), and treated with DNase I
(Ambion). 100 ng of total RNA, spiked with Poly-A controls, was used to synthesize cDNA, according to recommended protocols using the Ambion WT Expression kit. Fragmentation and labeling of cDNA was performed as per the Affymetrix GeneChip WT Terminal Labeling standard protocol. Samples were hybridized to Affymetrix Human Exon 1.0ST arrays, washed, stained and scanned with the Affymetrix GCS 3000 7G and GeneChip Operating Software v1.3 to produce
.CEL intensity files. QC analysis of all chips was performed with the Affymetrix expression console.
All array data was analyzed using tools in the Partek Genomic Suite of software (Partek
Inc., St. Louis, MO) [171]. Exon-level data was imported and filtered to include only those probes that are in the ‘Core Meta-probeset’, which represents 17,800 RefSeq genes and full-length
GenBank mRNAs. A pre-background adjustment was performed for GC content followed by
Robust Multi-Array Analysis (RMA) background correction, quantile normalization and mean probeset summarization [172, 173] (Supplementary Figure 1.27). Sources of variation due to random experimental factors, such as scan date and experiment were batch removed using
Analysis of Variance (ANOVA). The fold change and p-values for differentially expressed genes between ∆55k/∆ORF3 versus mock, ∆55k versus mock, ∆55k/∆ORF3 versus ∆55k and nutlin
versus mock were determined using linear contrasts in a 1-way ANOVA model using Method of
Moments [171]. The ANOVA model used was:
Yij = μ + Virus/Treatmenti + εij,
Yij represents the j-th observation on the i-th Virus/Treatment, μ is the common effect for the
whole experiment and εij represents the random error present in the j-th observation on the i-th
Virus/Treatment. The errors εij are assumed to be normally and independently distributed with
mean 0 and standard deviation δ for all measurements. A step-up false discovery rate (FDR) of 42
0.05 was applied to p-values calculated by ANOVA as the cut-off for significant differentially expressed genes.
Differentially expressed genes were analyzed using the Genomatix Pathway System
(GePS) and GeneRanker programs, which uses information extracted from public and proprietary databases. Overrepresentation of different biological terms (Gene Ontology categories, signal transduction pathways) within the input gene list are calculated and listed together with their respective p-value [174]. Signal Transduction Pathway Associations are obtained by Genomatix with a proprietary literature data mining algorithm based on all available Pubmed abstracts.
Promoter analysis.
Genomatix Gene2Promoter was used to retrieve the optimized promoters of the 265 differentially upregulated transcripts in ∆55k/∆ORF3 versus ∆55k infected SAECs and filter them for p53 TFBSs using MatInspector [175]. MatInspector searches transcription factor matrix matches based on position weight matrices [176], which has been used successfully to detect functional p53 transcription factor elements [177]. Six different matrices for p53 were used as described in Supplementary Table IV. Default matrix indices (core similarity: 0.75; matrix similarity: optimized) were set during TFBS searching. The "core sequence" of a matrix is defined as the (usually 4) consecutive highest conserved positions of the matrix. The maximum core similarity of 1.0 is only reached when the highest conserved bases of a matrix match exactly in the sequence. The matrix similarity score takes into account all bases over the whole matrix length. A perfect match gets a score of 1.00, a "good" match to the matrix usually has a similarity of >0.80.
Supplementary Data All supplementary data for Chapter 1 can be found published at: http://www.nature.com/nature/journal/v466/n7310/full/nature09307.html
Author Contributions Conrado Soria (C.S.) and Fanny Estermann (F.E.E.) contributed equally to this work.
C.S. performed the p53 activation and virus studies, including immunoblotting, RT-QPCR and microarray experiments. F.E.E. performed all chromatin immunoprecipitation and 43
immunofluoresence studies. K.C.E. performed the luciferase assays, E4-ORF3 sufficiency and complementation experiments, and assisted C.S with viral mutant studies. Clodagh O’Shea
(C.C.O.S.) analyzed the array data and wrote the paper with contributions from all authors.
C.C.O.S. was responsible for the overall conceptual design and supervision of the studies.
These data are published in Nature, August, 2010 entitled “Heterochromatin silencing of p53 target genes by a small viral protein”.
Chapter 2. p53 inactivation by oligomeric E4-ORF3 is specific to seroprevalent subgroup C adenoviruses
Abstract
The realization of a p53 selective oncolytic virus relies on the engineering of a virus capable of potent infection, replication and p53 selectivity of human tissues. Adenovirus encodes two proteins that inactivate the p53 pathway. E1B-55K binds and causes degradation of p53 whereas E4-ORF3 prevents access of p53 to its target gene promoters by induces de novo
heterochromatin formation at p53 target genes. Deletion of both E1B-55K and E4-ORF3
produces a virus that is defective for replication. A mutation in E1B-55K has already been
identified to separate its functions in p53 disruption and its other functions in viral replication.
Therefore, it is imperative to reveal the nature of E4-ORF3’s ability to inactivate p53 target genes
and if this function can be uncoupled from its other functions in viral replication to create a truly
selective p53 oncolytic adenovirus. Thus far, only E4-ORF3 from Subgroup C adenovirus type 5
has been shown to inactivate p53 target genes while it is unknown if E4-ORF3 from disparate
adenovirus subgroups also possess this function. To investigate this, we engineered hybrid
adenoviruses in which the Ad5 E4-ORF3 gene was swapped with E4-ORF3 genes from Ad9,
Ad12 or Ad34 in a ∆E1B-55K Ad5 background. We reveal that infection with ∆E1B-55K viruses
expressing Ad9, 12 or 34 E4-ORF3 genes were not able to prevent p53 target gene activation,
nor were those viruses able to replicate. However, E4-ORF3 from Ad9, 12 and 34 do mislocalize
TRIM24, TRIM33 and PML illustrating that targeting of these proteins is not sufficient for p53
target gene inactivation or viral replication. Furthermore, we show that MRN mislocalization is
exclusive to Ad5 E4-ORF3 suggesting that p53 inactivation and/or MRN mislocalization is
required for ∆E1B-55K Ad5 to replicate. Finally, we reveal residues in Ad5 E4-ORF3 that are
likely to contribute to functions specific to E4-ORF3 from Ad5 and may be mutated to separate its
functions in p53 inactivation and viral replication from those required for adenovirus replication.
44 45
Introduction
The study of viruses dates back to almost a century ago with the discovery of the tobacco mosaic virus [178]. Now, we have the ability to exploit human viruses for use as natural vessels for gene delivery or targeted cellular destruction [179, 180]. The more we know about viruses and the mechanisms by which they function, the more we will know how to engineer them to develop more sophisticated and targeted treatments. The Adenovirus has a 36kb genome that can be manipulated as a tool for use in research, gene delivery and oncolytics [181]. The eleven early expressed proteins encoded by the adenovirus are able to transform a quiescent cell into a virus production factory where DNA replication is initiated, apoptosis is blocked and anti-viral signals are inhibited [134]. Uncovering the precise residues and mechanisms by which early adenovirus proteins function is key to engineer novel targeted therapies.
E4-ORF3 is a 116 amino acid early adenovirus protein that oligomerizes to form wide nuclear cables with irregular thickness and branching. The crystal structure of E4-ORF3 revealed that E4-ORF3 was in fact a structured molecule and that its minor unit is a dimer held together through a hydrophobic beta-barrel interface [38]. E4-ORF3’s most unique feature is its method of oligomerization where the C-terminus of E4-ORF3 is able to pivot through a flexible Gly-Gly hinge and interact with other E4-ORF3 dimers via domain swapping allowing E4-ORF3 fibers to grow and branch in any direction. Using this approach, E4-ORF3 is able to build an insoluble nuclear avidity surface that inactivates five tumor suppressors simultaneously. The way in which E4-
ORF3 blocks the activation of p53 target genes is also extremely unique. Expressed early after adenovirus infection, E4-ORF3 triggers de novo heterochromatin formation specifically at p53 target genes denying access to high levels of phosphorylated p53 [37]. The mechanism E4-
ORF3 uses to induce heterochromatinization is unknown; however, this study aims to determine if nature can assist in solving this mystery.
The p53 protein was originally discovered by its binding to a DNA virus protein, and has since been found to be mutated in the majority of cancers [182]. The importance of p53 inactivation in DNA virus infection is evident in the fact that all DNA viruses encode proteins to 46
block p53 activation [183, 184]. Adenovirus E1B-55K mimics MDM2 and binds to the transactivation domain of p53, initiating degradation and blocking p53 transcriptional activation
[185]. On the basis that E1B-55K is required for p53 inactivation in adenovirus infected cells,
ONYX-015, an E1B-55K deleted virus was created as a p53 selective oncolytic therapy [186].
Unfortunately, it was impossible for ONYX-015 to be selective for p53, as p53 target genes were inaccessible due to heterochromatinization by E4-ORF3. E1B-55K and E4-ORF3 must both be deleted for adenovirus to activate p53 target genes, however, deletion of both genes results in a defective adenovirus that is unable to replicate [37]. Therefore, understanding which E4-ORF3 functions are critical for viral replication and what structures of E4-ORF3 are responsible for its multiple functions are important for the development of any adenovirus used as a human therapy.
Here, we compare E4-ORF3 proteins from disparate adenovirus subgroups examining conservation of functions and protein sequence to better understand the way in which E4-ORF3 functions.
There are 68 distinct serotypes of human adenoviruses divided into seven subgroups (A-
G) based on sequence homology [187-189], biophysical and biochemical criteria [190, 191]. All human adenoviruses encode E4-ORF3. Adenovirus serotype 5 (Ad5) of group C is the most well studied adenovirus serotype due to its high seroprevalence in humans [192, 193] and historical use in the creation of viral vectors for gene delivery [194, 195]. Ad5 is also the virus used in the discovery that E4-ORF3 inactivates p53 target genes [37]. Comparing and contrasting the conserved and non-conserved functions and sequences of disparate E4-ORF3 proteins will reveal the precise residues required for E4-ORF3’s many functions. In this study, we use Ad5 as a backbone in creating hybrid adenovirus species where the early protein E4-ORF3 gene from
Ad5 is swapped with E4-ORF3 genes from disparate adenovirus serotypes. This strategy allows us to study the properties of E4-ORF3 proteins from disparate adenovirus serotypes in the environment of the most well studied adenovirus, Ad5, infection.
Aside from its function in blocking p53 target gene activation, E4-ORF3 also targets the
Promyelocytic Leukemia protein (PML) [40]. The E4-ORF3 avidity surface attracts PML which is 47
then mislocalized from discrete nuclear bodies to E4-ORF3 cables. PML is mutated in Acute
Promyelocytic Leukemia where the N-terminus of PML is fused to the C-terminus of the Retinoic-
Acid Receptor-α due to a chromosomal translocation [196, 197]. The resulting fusion protein is thought to result in a gain of function for PML that progresses the leukemia forward [198, 199].
Although the precise function of PML is not known, it has been shown to be involved in a plethora of cellular functions including chromosome positioning [200], transcriptional activation [201], regulation of RNA [202], and activation of p53 [203]. PML has been shown to be required for acetylation of p53 [204] and can act as a transcriptional co-activator with p53 at p53 target genes
[205]. In addition, the deletion of PML in primary cells impairs p53 activation and inhibits apoptosis [206].
TRIM24 and TRIM33 are E4-ORF3 targets [41, 42] that belong to the large family of
Tripartite Motif proteins, of which PML is also a member (known as TRIM19). TRIM24 and
TRIM33 share structural and sequence similarity, each composed of a RING domain at the N- terminus, followed by two B-box domains a Coiled-coil and PHD and Bromo domains at the C- terminus. Through the PHD and Bromo domains, TRIM24 and TRIM33 have the ability to bind directly to histones with specific tail signatures [70, 81]. Each is also a transcription intermediary factor where TRIM24 regulates transcription initiated by nuclear receptors [207-210] and TRIM33 regulates functions of the SMAD family to regulate TGF-β transcription [109, 211, 212]. Similar to
PML, TRIM24 is also involved in translocations resulting in fusion genes that lead to cancer [103-
105] and TRIM33 is able to cooperate with Kras in mouse models to induce pancreatic tumors
[213]. Additionally, TRIM24 acts as an E3 Ub Ligase targeting p53 for ubiquitination and subsequent degradation [115]. These data illustrate the critical roles of TRIM24 and TRIM33 in regulating cell proliferation and apoptosis. Early in adenovirus infection, E4-ORF3 mislocalizes
TRIM24 and TRIM33 from a nuclear diffuse pattern to co-localization on E4-ORF3 nuclear tracks
[42]. Moreover, E4-ORF3 causes the diminishment of TRIM33 protein levels over the time course of infection [214]. However, it is unknown whether TRIM24 or TRIM33 play a role in the heterochromatinization of p53 target genes induced by E4-ORF3. 48
PML, TRIM24 and TRIM33 have been shown to be targeted by multiple E4-ORF3 proteins from disparate subgroups [41, 214-216], however, targeting the DNA damage signaling complex composed of Mre11, Nbs1 and Rad50 (MRN) is a function specific to Ad5 E4-ORF3
[215]. The MRN complex senses double strand DNA breaks and initiates a signaling cascade that results in the phosphorylation of p53 and subsequent cell cycle arrest [217]. E4-ORF3 immobilizes the MRN complex in nuclear tracks, preventing the MRN complex from initiating concatenation of adenovirus genomes [218, 219]. It is important to categorize conserved versus non-conserved traits of disparate E4-ORF3 proteins to understand how E4-ORF3 induces heterochromatin at p53 target genes and achieves its additional functions simultaneously.
Here we show that E4-ORF3 proteins from Subgroup A, Subgroup B and Subgroup D do not inactivate p53 target genes, revealing that this function is specific to Subgroup C adenoviruses. We engineered hybrid adenoviruses in which the Ad5 E4-ORF3 gene was swapped with E4-ORF3 genes from Ad9, Ad12 or Ad34 in a ∆E1B-55K Ad5 background. This method allowed us to study the functions of E4-ORF3 proteins within an established adenovirus transformed cellular environment. We found that infection with ∆E1B-55K viruses expressing
Ad9, 12 or 34 E4-ORF3 genes were not able to prevent p53 target gene activation. Furthermore, viral replication read out by adenovirus late protein expression was nullified in the hybrid viruses indicating that Subgroup C E4-ORF3 proteins have acquired functions required for ∆E1B-55K
Ad5 replication that E4-ORF3 proteins from Subgroups A, B and D do not possess. We also show that E4-ORF3 proteins from Subgroups A, B, C and D expressed by hybrid ∆E1B-55K viruses have conserved functions in mislocalization of PML, TRIM24 and TRIM33, and that MRN mislocalization is exclusive to Ad5 E4-ORF3. These results suggest that p53 inactivation and/or
MRN mislocalization is required for ∆E1B-55K Ad5 to replicate. These data also suggest that
MRN mislocalization and p53 inactivation by Ad5 E4-ORF3 are linked. Upon comparison of E4-
ORF3 protein sequences from Ad5, Ad9, Ad12 and Ad34, we reveal residues in Ad5 E4-ORF3 that are likely to contribute to functions specific to E4-ORF3 from Ad5. Furthermore, we identify residues likely to separate the ability of E4-ORF3 to mislocalize PML, TRIM24 and TRIM33, from 49
mislocalization of the MRN complex and p53 target gene inactivation which is valuable knowledge when engineering a p53 selective oncolytic adenovirus.
Results
E4-ORF3 sequence comparison among adenovirus subgroups.
Human adenoviruses are comprised of 68 members that fall into 7 distinct subclasses, A-
G. All human adenoviruses encode E4-ORF3 illustrating its importance in survival of the adenovirus throughout its evolution with humans. Previous analysis of the E4-ORF3 protein sequence has shown that E4-ORF3 proteins from subgroup C have diverged substantially from those of other human adenovirus species. To illustrate the divergence of Subgroup C E4-ORF3 proteins, we generated a rooted phylogenetic tree in which simian adenovirus E4-ORF3 sequences were set as the ancestral outgroup (Figure 2.1A). The horizontal branch length represents the number of amino acid differences between subgroups correlating to the evolutionary divergence. We see that the branch length of subgroup C adenoviruses is significantly longer than any other subgroup branch showing that E4-ORF3 proteins only from
Subgroup C have diverged in sequence which suggests a similar divergence in function. The divergence of subgroup C E4-ORF3 proteins is not shared with E1B-55K, whose functions in targeting the MRN complex and p53 activation overlap with E4-ORF3 (Figure 2.1B).
Furthermore, we show that in adenovirus Subgroup C, the pre-terminal protein that is essential for adenovirus replication, does not diverge significantly from the other adenovirus subgroups as seen with E4-ORF3 (Figure 2.1C). These data suggest that the sequence, structure, and function of E4-ORF3 from Subgroup C adenoviruses differ significantly compared to E4-ORF3 from all other adenovirus subgroups. Thus far, E4-ORF3 Subgroup C proteins are known to have a distinct function in the sequestration of the MRN complex, not observed in any other subgroup.
To investigate whether p53 target gene inactivation is also a function exclusive to E4-ORF3 from
Subgroup C adenoviruses, we chose to study E4-ORF3 proteins from the largest adenovirus subgroups, A, B, C and D. E4-ORF3 proteins from Ad12 from Subgroup A, Ad34 from Subgroup
B and Ad9 from Subgroup D were chosen to compare with E4-ORF3 from Ad5 Subgroup C. 50
E4-ORF3 amino acid sequences from Ad5, 9, 12 and 34 share 37.6% sequence identity (or
55.2% including semi-conserved residues) (Figure 2.1D). In contrast, E4-ORF3 from Ad9, 12 and
34 share 55.4% identity sequence (or 71.6% including semi-conserved residues) illustrating the considerable divergence of E4-ORF3 Ad5 from the other adenovirus serotypes. The divergence in amino acid sequence of Ad5 E4-ORF3 from the other subgroups gives us a map of amino acids most likely responsible for the acquired functions of E4-ORF3 from Subgroup C. Whether
E4-ORF3 proteins from Ad9, 12 or 34 do, or do not inactivate p53 target genes, this knowledge will facilitate the discovery of the precise mechanisms by which Ad5 E4-ORF3 functions in inactivating multiple tumor suppressors simultaneously.
Engineering and validation of mutant Ad5 adenoviruses.
To examine whether E4-ORF3 from Subgroup C adenoviruses have evolved a new function in p53 target gene inactivation, or if E4-ORF3 from disparate subgroups also block p53 activation, we engineered hybrid adenoviruses in which E4-ORF3 genes from Ad9 Subgroup D,
Ad12 Subgroup A, and Ad34 Subgroup B are swapped with E4-ORF3 Ad5 in an Ad5 Subgroup C background. Subgroup C viruses are the most well studied adenoviruses and therefore provide a predictable background to investigate the function of E4-ORF3. For Ad5 infection to activate p53 target genes, both E4-ORF3 and E1B-55K need to be deleted, as E1B-55K targets p53 for degradation. Therefore, to examine E4-ORF3’s function in inactivating p53 target genes, E4-
ORF3 genes from Ad9, Ad12 or Ad34 were engineered into an Ad5 ∆E1B-55K background.
Previous studies have used E1B-55K mutant adenoviruses, WtD, dl1520/ONYX-015 (E1B-55K deleted) and dl3112 (E1B-55K and ORF3 deleted) that were created from the parental adenovirus dl309, an Ad5 genome with significant deletions in the E3 region (detailed in Figure 51
Figure 2.1 Subgroup C E4-ORF3 protein sequences diverge from all other adenovirus subgroups.
Phylogenetic Trees based on the alignment of A. E4-ORF3 B. E1B-55K or C. pre-Terminal protein sequences from 66 distinct adenovirus serotypes. Height of subgroup triangles is proportional to the number of adenovirus serotypes within each subgroup. Protein sequences from simian adenoviruses were set as the outgroup for each tree. Horizontal branch lengths correspond to the number of amino acid differences between subgroups as indicated by the scale bar. D. Alignment of E4-ORF3 protein sequences from Subgroup A - Ad12, Subgroup B – Ad34, Subgroup C – Ad5 and Subgroup D – Ad9. Red and Green letters denote conserved and semi-conserved residues, respectively. 52
2.2A). Additionally, WtD, dl1520 and dl3112 contain E1A and E1B genes from adenovirus type 2 in place of Ad5 E1A and E1B genes. To investigate the function of E4-ORF3 in a clean genetic system, we engineered new deletion mutant adenoviruses. From the WT Ad5 genome, we generated mutants where expression of E1B-55K and/or E4-ORF3 are prevented, ∆E1B-55K
(∆55K) and ∆E1B-55K/∆E4-ORF3 (∆55K/∆ORF3) (detailed in Figure 2.2B). We also engineered a mock virus where the E1 region and E4-ORF3 gene is deleted to ensure that the virus does not initiate replication or express any E4-ORF3 that may blur our results. To determine if the WT,
∆55K and ∆55K/∆ORF3 viruses behave similarly to WtD, dl1520 and dl3112, we infected A549 cells (Figure 2.2C). We found that late viral proteins are similarly expressed over the time course of infection for all new and established viruses. Furthermore, p21 and MDM2 protein levels are suppressed in WT, ∆55K, WtD and dl1520 as compared to ∆55K/∆ORF3 and dl3112 infections where p21 and MDM2 levels are increased compared to mock infection. These results corroborate results found in previous studies and allowed us to generate hybrid viruses using
∆55K as a clean background for the engineering of hybrid viruses expressing E4-ORF3 from Ad5,
Ad9, Ad12 or Ad34.
To investigate if Ad9, Ad12 and Ad34 proteins are indistinguishable from Ad5 E4-ORF3 in preventing p53 transcriptional activation of downstream targets, we engineered hybrid adenovirus genomes using Ad5 ∆55K as a backbone and replaced the Ad5 E4-ORF3 gene
sequence with the sequences from Ad9, Ad12 or Ad34 E4-ORF3 genes (detailed in Figure 2.3A).
Even though there are no antibodies that recognize Ad9, Ad12 or Ad34 E4-ORF3 proteins, we did
not tag the proteins due to concerns in disruption of E4-ORF3 function upon incorporation of a tag
into its structure. Thus, we infected A549 cells with the mock, WT, ∆55K, ∆55K/∆ORF3,
∆55K/ORF3-Ad9, ∆55K/ORF3-Ad12, and ∆55K/ORF3-Ad34 viruses and analyzed lysates by
western blot after 24 hours post infection (h.p.i.) (Figure 2.3B). The E2A adenovirus protein is
expressed throughout viral infection and is required for the creation of viral replication domains.
We see that E2A is expressed during all viral infections except for the mock virus illustrating that
the hybrid viruses are indeed able to infect A549 cells and initiate transcription of E2A. 53
Furthermore, we show that E1B-55K is not expressed in ∆55K, ∆55K/∆ORF3, ∆55K/ORF3-Ad9,
∆55K/ORF3-Ad12, or ∆55K/ORF3-Ad34 infections.
Serotype-specific inactivation of p53 target genes by E4-ORF3.
To test if p53 target gene inactivation by E4-ORF3 is specific to Ad5, we infected A549
cells with mock, WT, ∆55K, ∆55K/∆ORF3, ∆55K/ORF3-Ad9, ∆55K/ ORF3-Ad12 and ∆55K/ORF3-
Ad34 viruses and analyzed p53 target genes over the time course of infection. MDM2 and p21
are well characterized p53 target genes, activated during ∆55K/∆ORF3 infection. Here we show
that MDM2 and p21 levels are suppressed by WT and ∆55K infection as compared to uninfected
and mock (Figure 2.4A). Conversely, during infection with ∆55K/∆ORF3 and ∆55K hybrid viruses
expressing E4-ORF3 from Ad9, 12 and 34, MDM2 and p21 levels are high compared to mock,
indicating that p53 target gene inactivation is specific to Ad5 E4-ORF3. Additionally, we show
that mRNA levels of p21 and MDM2 mirror the changes seen in protein levels and also
demonstrate that additional p53 transcriptional targets PUMA, GADD45A, CCNG and 14-3-3σ
also follow the same trend as p21 and MDM2 (Figure 2.4B).
In addition, we demonstrate that WT and ∆55K infected lysates accumulate similar levels
of late viral proteins, a reliable indicator of viral replication, over the time course of viral infection.
However, infection with ∆55K/ORF3-Ad9, ∆55K/ORF3-Ad12 or ∆55K/ORF3-Ad34 shows a
complete absence of late viral proteins equivalent to that seen in ∆55K/∆ORF3 infection (Figure
2.4A). Taken together, these data demonstrate that the ability of E4-ORF3 to inactivate p53
target genes and support viral replication are functions exclusive to subgroup C adenoviruses.
This result also demonstrates that there are natural mutations in E4-ORF3 that give Subgroup C
E4-ORF3 proteins the ability to block the activation of p53 target genes.
54
Figure 2.2 Engineering and validation of ∆E1B-55K and ∆E1B-55K/∆E4-ORF3 mutant adenoviruses.
A. Established mutations in the E1, E2, E3 and E4 regions of WtD, dl1520 (ONYX-015) and dl3112 historical mutant adenoviruses. B. Mutations in new viruses WT (PCMN-102), ∆E1B- 55K (PCMN-124), ∆E1B-55K/∆E4-ORF3 (PCMN-140), and mock (PCMN-117). C. Western blot of lysates from A549 cells uninfected (UN) or infected with the indicated viruses and harvested at 24, 36 or 48 hours post infection (h.p.i.). Western blot was probed with antibodies against p53, MDM2, p21, Adenovirus type 5 E4-ORF3, E1B-55K and Late viral proteins (Hexon, Penton and Fiber). 55
Figure 2.3 Engineering and validation of ∆E1B-55K mutant adenovirus with Ad9, Ad12 or Ad34 E4-ORF3 genes replacing E4-ORF3 from Ad5.
A. Mutations in the E1, E2, E3 and E4 regions of adenovirus mutants ∆E1B-55K/E4-ORF3- Ad9 (PCMN-162), ∆E1B-55K/E4-ORF3-Ad12 (PCMN-163) and ∆E1B-55K/E4-ORF3-Ad34 (PCMN-164). B. Western blot of lysates from A549 cells uninfected (UN) or infected with PCMN-117 (mock), PCMN-102 (WT), PCMN-124 (∆55K), PCMN-140 (∆55K/∆ORF3), PCMN- 162 (∆55K/ORF3-Ad9), PCMN-163 (∆55K/ORF3-Ad12 and PCMN-164 (∆55K/ORF3-Ad34), harvested at 24 h.p.i. and probed for Adenovirus type 5 E2A, E4-ORF3, E1B-55K and β-actin as a loading control. 56
E4-ORF3 proteins that do not inactivate p53 target genes or complement ∆E1B-55K replication do mislocalize PML, TRIM24 and TRIM33.
To understand the mechanism behind Ad5 E4-ORF3 p53 target gene inactivation, it is critical to investigate its known targets. E4-ORF3 proteins from Subgroup A, C and E have been shown to mislocalize PML into nuclear track-like structures that co-localize with E4-ORF3 [215,
216]. PML has also been shown to have a role in p53 activation and chromatin looping. Seeing as E4-ORF3 proteins from Ad9, Ad12 and Ad34 do not block p53 target gene activation, or complement Ad5 ∆E1B-55K replication, we tested if E4-ORF3 proteins from hybrid viruses encoding Ad9, Ad12 and Ad34 E4-ORF3 proteins could target PML. Therefore, we infected A549 cells with ∆E1B-55K viruses expressing E4-ORF3 from Ad5, Ad9, Ad12 or Ad34 and probed for endogenous PML (Figure 2.5A). The antibody to Ad5 E4-ORF3 does not cross-react with Ad9,
12 or 34 E4-ORF3 proteins. Therefore, to identify infected cells, we co-stained cells with early viral protein E2A. We show that Ad9, Ad12 and Ad34 E4-ORF3 proteins phenocopy E4-ORF3 from Ad5 in mislocalizing PML into nuclear tracks, as compared to uninfected or ∆55K/∆ORF3
where PML is retained in bodies. This result indicates that mislocalization of PML is not sufficient
for E4-ORF3 to inactivate p53 target genes.
In addition to PML, E4-ORF3 has been shown to bind and mislocalize TRIM24 and
TRIM33 into irregular nuclear fibers . TRIM24 regulates nuclear receptor transcription and also
instigates degradation of p53 while TRIM33 regulates the TGF-β pathway. To test if TRIM24 or
TRIM33 are targeted by E4- ORF3 in non-replicating, p53 activating hybrid viruses, we infected
cells with ∆55K, ∆55K/∆ORF3, ∆55K/ORF3-Ad9, ∆55K/ORF3-Ad12, and ∆55K/ORF3-Ad34
viruses. Unfortunately, we are unable to detect endogenous TRIM24 or TRIM33 co-localization
with Ad5 E4-ORF3 using our current antibodies (see methods). Therefore, we expressed GFP
tagged TRIM24 and TRIM33 in easily transfectable U2OS cells and subsequently infected with
the aforementioned viruses (Figure 2.5B and 6C). GFP-TRIM24 and GFP-TRIM33 are 57
Figure 2.4 Ad9, Ad12 and Ad34 E4-ORF3 proteins do not suppress activation of p53 transcriptional targets or support viral replication.
A. Western blots of A549 cells uninfected (UN) or infected with indicated viruses harvested at 24 and 36 h.p.i. probed with antibodies against p53 and p53 targets MDM2and p21. Also probed with antibodies against Ad5 viral proteins, E4-ORF3 and E1B-55K. β-actin probed as a loading control. B. Q-PCR results of p53 target genes p21, MDM2, PUMA, GADD45A, CCNG and 14-3-3σ from A549 cells infected with indicated viruses harvested at 24 and 36 h.p.i. shown as fold change relative to uninfected (UN). C. Same conditions as (A.) probed with adenovirus type 5 late viral proteins. 58
nuclear diffuse in cells uninfected or infected with ∆55K/∆ORF3. GFP-TRIM24 is mislocalized onto E4-ORF3 nuclear tracks, similar to and marked by mislocalized PML when cells are infected with viruses expressing Ad5, Ad9, Ad12 or Ad34 E4-ORF3 genes. GFP-TRIM33 is also mislocalized into nuclear tracks, however GFP-TRIM33 mirrors PML localization, whereas GFP-
TRIM24 is localized adjacent to PML on E4-ORF3 tracks. Nevertheless, these data indicates that
TRIM24 and TRIM33 mislocalization by E4-ORF3 is conserved among disparate subgroups, and that their mislocalization is not sufficient for E4-ORF3 to complement a ∆E1B-55K Ad5 virus in p53 inactivation or viral replication.
In addition to the mislocalization of TRIM33, E4-ORF3 also causes the dramatic decrease of
TRIM33 protein levels during Ad5 or Ad12 infection [214]. This decrease in TRIM33 protein was inhibited by the addition of a proteosome inhibitor suggesting that E4-ORF3 has a role in the degradation of TRIM33. To test if Ad9, Ad12, or Ad34 E4-ORF3 proteins expressed by hybrid viruses are able to initiate a decrease in TRIM33 protein levels, we probed lysates of infected
A549 viruses with an antibody to endogenous TRIM33 (Figure 2.5D). Immunoblot analysis reveals that in a ∆E1B-55K Ad5 background, E4-ORF3 from Ad5, Ad9 and Ad34 are able to decrease TRIM33 protein levels by 36 h.p.i., as compared to Ad12 E4-ORF3 which did not complement ∆E1B-55K in reduction of TRIM33. Interestingly, TRIM33 protein reduction varies among hybrid viruses where infection with ∆55K/ORF3-Ad9 greatly diminishes TRIM33 levels by
24 h.p.i. whereas viruses expressing Ad5 or Ad34 genes do not significantly decrease TRIM33 levels until 36 or 48 h.p.i. as compared to mock infection. Seeing as Ad12 E4-ORF3 was shown to be able to decrease TRIM33 upon transfection in HeLa cells, it is likely that Ad12 E4-ORF3 has altered interactions with proteins in A549 cells infected with ∆55K/ORF3-Ad12 hybrid virus.
However, these data reveal that a drop in TRIM33 levels caused by E4-ORF3, along with PML,
TRIM24 and TRIM33 mislocalization are not sufficient for E4-ORF3 to inactivate p53 target genes or complement a ∆E1B-55K deleted Ad5 virus in replication.
59
Figure 2.5 Mislocalization of PML, TRIM24 and TRIM33 is a conserved function of E4- ORF3 among disparate adenovirus subgroups and is not sufficient to inactivate p53 target genes.
A. A549 cells were infected with the indicated viruses, fixed 24 h.p.i., and analyzed by immunofluorescence for subcellular localization of E4-ORF3-Ad5 (white), PML (red), Ad5 E2A (green) and DNA (blue). U2OS cells were transfected with B. GFP-TRIM24 or C. FLAG- TRIM33. 4 hours post transfection, cells were infected with the indicated viruses. 24 h.p.i. cells were fixed and analyzed by immunofluorescence for E4-ORF3-Ad5 (white), PML (red), and GFP-TRIM24 (green) or FLAG-TRIM33 (green). Nuclei are indicated with blue line in merged images. D. A549 cells were infected with the indicated viruses and harvested at the indicated times post infection. Protein lysates were analyzed for endogenous TRIM33 with β- actin blotted as a loading control. 60
Figure 2.6 - The MRN complex is only mislocalized in cells infected with viruses expressing Ad5 E4-ORF3 that are able to replicate and inactivate p53. A. A549 cells were infected with the indicated viruses, fixed after 24 h.p.i. and analyzed by immunofluorescence for E4-ORF3-Ad5 (white), PML (red) and Mre11 (green) or B. Nbs1 (green). Nuclei are indicated with blue line in merged images. 61
MRN sequestration and p53 target gene inactivation are functions specific to E4-ORF3 from Subgroup C adenoviruses
Mislocalization of the MRN DNA damage sensing complex is a function specific to
subgroup C E4-ORF3 proteins. To verify that in our system, the MRN complex is mislocalized in
an E4-ORF3 subgroup C specific manner, we stained infected A549 cells with antibodies to
endogenous Mre11 or Nbs1 and PML as a tracker for E4-ORF3 localization (Figure 2.6A and B).
We observe that Mre11 and Nbs1 are not mislocalized upon infection with ∆E1B-55K hybrid
viruses expressing Ad9, Ad12 or Ad34 E4-ORF3 genes. Conversely, Ad5 E4-ORF3 mislocalizes
Mre11 and Nbs1 onto E4-ORF3 nuclear tracks compared to their nuclear diffuse subcellular
localization in uninfected or ∆55K/∆ORF3 infected cells. Furthermore, we observe that Mre11 is
degraded during WT Ad5 infection due to the expression of E1B-55K. Our data corroborate
previously published results in which MRN mislocalization is specific to E4-ORF3 from Subgroup
C. Taken together, we show that E4-ORF3 from Subgroup C adenoviruses acquired functions in
both MRN disruption and p53 target gene inactivation, whereas PML, TRIM24 and TRIM33
disruption are conserved across disparate adenoviruses. Therefore, we are able to compare E4-
ORF3 protein sequences among these species to elucidate the structure of Ad5 E4-ORF3
responsible for its acquired functions.
Comparison of sequence and structures of E4-ORF3 dimers.
Comparing the amino acid sequences of E4-ORF3 proteins from subgroup C to
subgroups unable to inactivate p53 target genes, we will be able to identify residues likely to
contribute to the conserved and non-conserved functions of E4-ORF3. The crystal structure of a
soluble mutant of E4-ORF3 was recently elucidated and shown to be a structured protein forming
a dimer unit [38]. We used the crystal structure of E4-ORF3 to identify residues whose side
chains contribute to the formation of the protomer core, the dimer interface, or are surface
exposed (Figure 2.7). To determine which E4-ORF3 residues are surface exposed, we subjected
the Ad5 E4-ORF3 crystal structure to analysis by the GETAREA program which calculates the
area of surface exposes side chains of each residue within E4-ORF3. In addition, we identify 62
residues that are conserved among E4-ORF3 proteins from Ad5, 9, 12 and 34 (shown in red), non-conserved residues (shown in black), and residues that are not conserved only in Ad5 E4-
ORF3 (shown in blue). Analysis of the crystal structure reveals that there are 25 residues that face the protomer core and contribute to the protomer structure (Fig.7A). Of these residues, 84% are conserved among all E4-ORF3 serotypes analyzed (Figure 2.7F). This high level of conservation suggests that the E4-ORF3 protomer structure is conserved among all subgroups.
Surprisingly, of the 12 residues that face the β-barrel interface, only 25% are conserved among the four E4-ORF3 serotypes analyzed (Figure 2.7B and F). This is extremely surprising given that the macromolecular structure of E4-ORF3 in the nucleus very similar among all E4-ORF3 proteins. The divergence of the residues within the β-barrel interface suggest that the 3D structure of E4-ORF3 dimers from Ad9, 12 and 34 are significantly different than that of Ad5 and may explain Ad5 E4-ORF3’s additional functions. Finally, we show that of the 46 solvent exposed residues of E4-ORF3, 48% are conserved among all adenovirus serotypes analyzed
(Figure 2.7C and F). 62% of E4-ORF3 solvent exposed residues are conserved among Ad9,
Ad12 and Ad34 illustrating the similarities among Subgroups A, B and D, not conserved in
Subgroup C. Taken together, these data suggest that the acquired functions of Subgroup C E4-
ORF3 proteins in inactivating p53 target genes and mislocalizing the MRN complex are not due to single point mutations on the surface of E4-ORF3, and instead paint a more complex picture.
Seeing as the surface exposed residues will undoubtedly have an impact on E4-ORF3 function, we further analyzed them and their placement on the E4-ORF3 surface (Figure 2.8). To illustrate which residues are conserved, non-conserved or not conserved only in Ad5 E4-ORF3, we color coded the surface exposed residues on the ribbon structure of the Ad5 E4-ORF3 dimer
(Figure 2.8C). Additionally, we show the space filled model of the color coded E4-ORF3 structure
from various angles (Figure 2.8D). It is intriguing to highlight the residues that are conserved 63
Figure 2.7 Residues of the β-barrel interface and solvent exposed residues of E4-ORF3 dimer are most different in comparison of E4-ORF3 amino acid sequences from disparate serotypes.
Alignment of E4-ORF3 amino acid sequences from Ad9-Subgroup D, Ad12-Subgroup A, Ad34-Subgroup D and Ad5-Subgroup C showing corresponding alpha helices or beta sheets shown in (D). A. Open carrot residues represent residues facing E4-ORF3 protomer core. B. Starred residues represent residues facing the beta-barrel interface between the two E4- ORF3 protomers that make up a dimer. C. Closed carrots represent residues that are solvent exposed. Residues highlighted in A.-C. were determined based the crystal structure of the N82 E4-ORF3 Ad5 dimer. D. Key for A.-C. where red letters represent residues conserved or semi-conserved among all E4-ORF3 serotypes shown, blue letters represent residues where E4-ORF3 from Ad5 differ from that of the other serotypes, and black represent residues non- conserved among all serotypes shown. E. Cartoon model of Ad5 E4-ORF3 N82A dimer, one protomer in grey and one in orange. Beta-sheets and alpha-helices of one protomer are labeled. F. Percentage of conserved and non-conserved residues that are facing the protomer core, beta-barrel interface or are solvent exposed. 64
Figure 2.8 The crystal structure of E4-ORF3-Ad5 reveals the similarities and differences among disparate E4-ORF3 proteins.
A. Comparison of E4-ORF3 amino acid sequences from Ad9, Ad12, Ad34 and Ad5. B. “Key for E4-ORF3 Residues” specifies the meaning of residue colors in (A-D). Grey rectangles underneath the alignment correspond to residues that make up the indicated beta-sheet or alpha-helices shown in (C) C. Cartoon model of Ad5 E4-ORF3 N82A dimer showing solvent exposed residues in colors coordinating to the key in (B). Beta-sheets and alpha-helices are labeled. D. Cartoon models of Ad5 E4-ORF3 N82A dimer at indicated angles above space filled models of the cartoon shown in (C). 65
among E4-ORF3 proteins from Ad9, 12 and 34 that are not conserved in Ad5 because these residues are what cause Ad5 E4-ORF3 to be divergent from all other adenovirus subgroups
(Figure 2.1A). These residues may also be responsible for Ad5 E4-ORF3’s acquired functions in mislocalizing the MRN complex and blocking p53 target gene activation. Additionally, analysis of the conserved surface exposed residues of E4-ORF3 will also reveal likely interaction sites for
conserved targets PML, TRIM24 and TRIM33. We observe that the loop structure connecting α2
with β2 combined with α3 is a region of high conservation (Figure 2.A and D side view). This site
is likely to be required for TRIM binding although we believe that multiple mutations would need
to be made in this domain to abolish an interaction. We also observe a highly variable region
among all E4-ORF3 proteins created by the loops connecting β2 and β3 (Figure 2.8C and D front
and bottom views). These residues flank a “keyhole” opening and residue differences in this area
will change the structure of the opening. The “keyhole” will also be altered by the changes within
the β-barrel interface causing the dimer to be held together more tightly, or loosely, and altering
the size of the opening. This variable region is a likely candidate to explain Ad5 E4-ORF3’s
acquired functions.
Discussion
The overlapping functions of E1B-55K and E4-ORF3 in Subgroup C adenoviruses was
unexpected and as such has complicated the creation of a p53 selective oncolytic virus. Seeing
as a ∆E1B-55K/∆E4-ORF3 mutant virus is unable to replicate, even in p53 defective cells, the
required functions of E4-ORF3 in viral replication need to be determined and separated from its
functions in blocking p53 target gene activation. Here we show that inactivation of p53 target
genes is a function specific to E4-ORF3 from Subgroup C adenoviruses, as is the mislocalization
of the MRN complex. We also show that E4-ORF3 proteins from Subgroups A, B and D are
unable to facilitate viral replication when expressed in hybrid Ad5 ∆E1B-55K viruses where E4-
ORF3 genes from Ad9, 12 and 34 are swapped with the Ad5 E4-ORF3 gene. However, E4-
ORF3 proteins expressed by the hybrid viruses are able to mislocalize PML, TRIM24 and
TRIM33 indicating that the disruption of these targets is insufficient for inactivation of p53 target 66
genes or for supporting viral replication in an Ad5 ∆E1B-55K background. This suggests that mislocalization of PML, TRIM24 and TRIM33 may not be required functions of E4-ORF3 to support first round viral replication and instead may serve to block anti-viral signaling. Evidence of this is supported by data showing that PML knock-down phenocopies E4-ORF3’s antagonization of interferon mediated inhibition of adenovirus replication [220]. Furthermore,
PML, TRIM24 and TRIM33 belong to a large family of Tripartite Motif (TRIM) proteins known as inhibitors of retrovirus replication [61]. It is likely that the anti-viral properties of the TRIM family is not solely linked to retroviral replication and instead may be a dynamic and general anti-viral set of proteins targeted by many viruses (which I show in Chapter 3).
On top of p53 pathway inactivation, Ad5 E4-ORF3 and E1B-55K both target the MRN complex. While E4-ORF3 mislocalizes MRN onto nuclear tracks, E1B-55K causes degradation of
Mre11 [221]. Consequently, when E1B-55K and E4-ORF3 are both deleted from Ad5, the MRN complex is uninhibited and may be blocking viral replication. Evidence for this hypothesis is given in the result that E4-ORF3 proteins from Ad9, 12 and 34 do not mislocalize the MRN complex, nor support viral replication in the absence of E1B-55K. This implicates the MRN complex as a critical target for E4-ORF3 to support adenovirus replication when E1B-55K is deleted. However, the targeting of MRN and p53 target genes by Ad5 E4-ORF3 may be linked and difficult to functionally separate by mutations in E4-ORF3.
The functional differences between Subgroup C E4-ORF3 proteins and E4-ORF3 from other adenovirus subgroups are certainly associated with residue changes that alter the protein shape and surface. Luckily, the dimer structure of a soluble mutant of Ad5 E4-ORF3 was recently published and allowed us to use the structure to determine the contribution of amino acids to the stabilization of the protomer core, β-barrel interface, and which are surface exposed.
The soluble mutant of E4-ORF3 (N82E) was also shown to preserve its function in mislocalizing
PML [38] indicating that the dimer structure elucidated is will have a preserved PML binding site.
Seeing as PML mislocalization is a conserved function among all E4-ORF3 proteins, it is
probable that the conserved solvent exposed amino acids are involved in the interaction. We 67
show that the most conserved solvent exposed region of E4-ORF3 is in the loop connecting α2 with β2 combined with α3. Within this region there are 10 conserved residues among all E4-
ORF3 proteins from disparate subgroups which is 45% of all solvent exposed conserved residues, the rest of which are not condensed to a single area. E4-ORF3 forms an avidity surface through higher order oligomerization which is required for all of its functions. The avidity surface allows E4-ORF3 to interact with proteins with very low affinity for one another. Therefore, we believe that multiple mutations would need to be made in the putative PML binding surface of E4-
ORF3 to completely abolish PML binding. Conversely, MRN is not mislocalized by the soluble
E4-ORF3 dimer suggesting a novel interface constructed upon higher order oligomerization.
Alternatively, precise nuclear localization may be required for MRN to be disrupted by E4-ORF3.
However, sequence analysis of E4-ORF3 proteins does reveal a highly variable region among
E4-ORF3 proteins created by the loops connecting β2 and β3. These residues flank a “keyhole” opening and residue differences in this area will change the structure of the opening. The
“keyhole” will also be altered by the changes within the β-barrel interface where only 25% of
amino acids are conserved among E4-ORF3 proteins from disparate subgroups. We believe that
this region may be required for precise E4-ORF3 nuclear localization and is required for
inactivation of p53 target genes. Furthermore, there are significant similarities and differences
within the C-terminal tail of E4-ORF3 proteins, however because we are unable to crystallize wild-
type E4-ORF3, we are unsure of the contribution of the tail to the structure of oligomerized E4-
ORF3. Nonetheless, we know that the C-terminal tail is incorporated into the β-barrel
dimerization interface. This inclusion will drastically alter the largest solvent exposed interface of
E4-ORF3 (Figure 2.8D, front view) and likely contribute to functional differences among E4-ORF3
proteins.
It is interesting to speculate why E4-ORF3 from Subgroup C has acquired overlapping
functions with E1B-55K in mislocalizing the MRN complex and inactivating the p53 pathway. One
striking attribute of Subgroup C adenoviruses is their high seroprevalence compared to other
subgroups in the human population around the world [43, 44, 222]. It is conceivable that the 68
acquired functions of E4-ORF3 in Subgroup C adenoviruses in blocking p53 target gene transcription and mislocalization of the MRN complex contributes to the success of these viruses.
MRN has been shown to inhibit viral replication in E4 deleted viruses and links viral genomes end to end using non-homologous end joining [218]. Perhaps early mislocalization of the MRN complex by E4-ORF3 allows for very low levels of infection to proceed into replication until E1B-
55K is expressed at high enough levels to degrade Mre11. Furthermore, 25% of the genes whose transcription is blocked by E4-ORF3 are not p53 regulated (not activated by Nutlin-3 or lacking a p53 binding site) [223]. Perhaps inactivation of p53 target genes is not the main function of E4-ORF3’s induction of heterochromatinization. Alternatively, at early times during infection, p53 may escape degradation by E1B-55K and activate transcription of genes that may signal to the immune system. The major contribution of E4-ORF3 functions in viral replication is not well understood and may not be obvious in cell culture experiments. Therefore, research of
E4-ORF3 in the context of the human immune system may be vital to discover its critical functions.
Materials and Methods
Phylogenetic Analysis
66 E4-ORF3, E1B-55K and pre-Terminal Protein sequences were obtained from NCBI from adenovirus genomes where the E4-ORF3 gene was defined (all adenovirus serotypes except Ad26, Ad48 and Ad49, IDs listed in Supp. Table 1). Protein sequences were aligned using ClustalW2 Multiple Sequence Alignment program [224]. Phylogenetic analysis of the aligned sequences was performed using the Neighbor-Joining Method, No. of amino acid differences substitution model, and pairwise deletion for gaps using the MEGA 5.0 software [225].
Cells
Human lung carcinoma cell line A549 and human bone osteosarcoma cell line U2OS cells were cultured in Dulbecco modified Eagle medium (DMEM, Invitrogen) supplemented with
10% (vol/vol) Fetal Calf Serum (FCS), kept at 37°C, in a humidified 5% CO2 atmosphere. Small 69
Airway Epithelial Cells (SAECs) were immortalized by constitutively expressed hTERT. SAECs were cultured with Small Airway Epithelial Cell Growth Medium (SABM) + SingleQuot supplements without gentamycin (Lonza) kept at 37°C, in a humidified 5% CO2 and 3% O2 environment..
Viruses
dl309, WtD, dl1520 (ONYX-015) and dl3112 are described previously in [226], [135], and
[227]. Mock (PCMN-118), WT (PCMN-102), ∆E1B-55K (PCMN-124) and ∆E1B-55K/∆E4-ORF3
(PCMN-140) ∆E1B-55K/E4-ORF3-Ad9 (PCMN-162), ∆E1B-55K/E4-ORF3-Ad12 (PCMN-163), and ∆E1B-55K/E4-ORF3-Ad34 (PCMN-164) is the same as ∆E1B-55K (PCMN-124) with the E4-
ORF3 gene replaced with E4-ORF3 sequences from Ad9, Ad12, or Ad34. Viruses were titered on 293/E4/pIX cells, as described previously in which E4 is under inducibe control of dexamethazone [36]. A549 cells and SAECs were infected with an MOI of 10 and U2OS cells with MOI 30.
Plasmids and transfection
TRIM33 was cloned into Gateway vector pENTR (Invitrogen). Via Gateway LR recombination [228], the TRIM33 gene was fused with the FLAG sequence in frame at the N- terminus with a DEST vector pcDNA3 backbone and expressed via CMV promoter. TRIM24 was cloned into Gateway vector pDONR221 (Invitrogen). Using gateway LR recombination, the
TRIM24 gene was fused with the GFP sequence in frame at the N-terminus using pDEST53
(Invitrogen) and expressed via CMV promoter. Plasmids were transfected into U2OS cells using
Lipofectamine 2000.
Western Blots
Cells were lysed using RIPA buffer (100mM Tris pH 7.4, 300mM NaCl, 2mM EDTA, 2%
Triton X-100, 2% Deoxycholic acid, 2mM NaF, 0.2mM NaVO4, 2mM DTT, 0.1% SDS) with mini- protease cocktail (Roche), 10mM NEM, 0.5mM PMSF, 10nM Calyculin A, 5uM TSA, 5mM
Sodium butyrate, and three rounds of 30s sonication with amplitude of 65. 50ug protein lysate was loaded per well in pre-cast 4-20% Tris-Glycine gels (Invitrogen). Proteins were transferred 70
using the Bio-Rad wet transfer system. Blots were blocked with 5% powdered milk in TNT (0.5M
TrisCl pH 8.0, 1.5M NaCl, 1% Tween20). Primary antibodies used for western blots: Ad5
(abcam), p53 (DO-1, Santa Cruz Biotech), MDM2 (2A10, Oncogene), p21 Cip1/Waf1 (Millipore),
Ad5 E1A (m73, Calbiochem), Ad5 E2A (B6-8, gift from Arnold Levine), Ad5 E4-ORF3 (Ascenion),
Ad5 E1B-55K (2A6, [229]), TRIM33 (Bethyl), Mre11 (Novus), Nbs1 (GeneTex), β-actin (abcam).
Primary antibodies were detected with secondary antibodies labeled with either IRDye800
(Rockland), or Alexa Fluor 680 (Molecular Probes). Fluorescent antibodies were visualized using
a LI-COR-Odyssey scanner.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 30min, permeabilized with .1% Triton X-
100 for 5min and blocked with 5% Normal Goat Serum (Jackson ImmunoResearch) in PBS for
20mins. Cells were incubated with primary antibodies for 1 hour and washed once for 1min and
twice for 20min. Cells were then incubated with secondary antibodies for 1 hour and washed
once for 1min and twice for 20min (Alexa Flour 488, 555, and 633 from Invitrogen). Slides were
then rinsed with diH2O and stained with Hoechst. Slides were mounted with ProLong Gold
(Invitrogen). Images were acquired using a Zeiss AxioPlan 2 microscope at 63X using AxioVision
4.6 software. Images were edited using programs ImageJ [230] and Photoshop (Adobe).
Primary antibodies used for IF: PML (PG-M3, Santa Cruz Biotech), PML (Bethyl), Mre11
(Genetex), Nbs1 (Novus), Ad5 E4-ORF3 (Ascenion), Ad5 E2A (B6-8, gift from Arnold Levine),
FLAG tag (Sigma).
RT-qPCR
Total RNA was harvested using the Ambion Purelink RNA mini kit according to
manufacturers’ instructions. 1ug of total RNA was reverse transcribed using qScript supermix
from Quanta bioscience which and diluted to 5ng/ul RNA input in H2O. 10uL qPCR reacations
were set up using Bio-Rad Soo Fast probes supermix with 10ng of input cDNA for 18s and 40ng
for all other targets. All samples were normalized to 18S. Samples were run on a Bio-Rad CFX96.
71
Author Contributions
Kristen Espantman performed all experiments in Chapter 2. Dr. Jason DeHart performed
Real time qPCR shown in Figures 2.4. I thank Dr. Jason DeHart of the O’Shea lab for processing and analyzing RNA expression levels using his expertise in qPCR in Chapter 2. I would like to thank Dr. Colin Powers of the O’Shea lab for developing a straightforward and fast method for engineering adenovirus mutants that allowed me to quickly create and validate E4-ORF3 adenovirus mutants used in this study. I thank Dr. Horng Der Ou for the analysis of E4-ORF3 solvent exposed residues and his constant advice regarding E4-ORF3 structure and function.
Chapter 3 – A subset of oligomeric TRIM proteins have conserved interactions with disparate DNA virus proteins
Abstract
The Promyelocytic Leukemia protein (PML) is targeted by tumor mutations and several
DNA virus proteins. However, unlike critical regulatory proteins p53 and Rb which are also converged upon by tumor mutations and viruses, PML was not shown to be a vital checkpoint protein in the regulation of cell growth or death. Why then is PML converged upon by multiple viral proteins? Here we show that viral proteins known to disrupt PML do so as part of a much larger strategy to disrupt a key subset of the Tripatite Motif (TRIM) family of proteins, of which
PML is a member. The TRIM family is composed of over 70 human proteins with conserved N- terminal domains and varied C-termini with each domain acting as a protein-protein interaction interface for homo- and hetero-oligomerization. We show that the TRIM interactome containing first order interacting proteins contains over 900 nodes (proteins) and 12,500 edges
(interactions). Analysis of the set of proteins in this protein network reveals the contribution of the
TRIM interactome in the regulation of many processes including cell cycle, apoptosis, and stress responses as well as the TGF-β, TNF-α, p53 and nuclear receptor pathways. Here, we reveal novel interactions of a subset of TRIM proteins with oligomeric DNA virus proteins E4-ORF3,
Herpes Simplex Virus-1 ICP0 and Cytomegalovirus IE2. All three viral proteins mislocalize or are mislocalized by TRIM15, 22, 24, 33 and 61. TRIM15 and 22 are potent inhibitors of retroviral infection, while TRIM33 regulates the TGF-β pathway and TRIM24 is a regulator of the Interferon and STAT signaling pathways. Moreover, we show that E4-ORF3 and ICP0 do not target a single TRIM domain, and instead exhibit interactions with multiple domains within TRIMs. Super- resolution microscopy reveals various interaction phenotypes of TRIMs with E4-ORF3 and ICP0.
A layering effect is observed for TRIM24 and 33 where TRIM24 lies on top of TRIM33 which is on top of E4-ORF3 cables whereas TRIM61 is observed within E4-ORF3 cables. Similarly, TRIM61 is localized on the inside of ICP0 nuclear rings, whereas TRIM27 is localized directly on top of
72 73
ICP0 rings. These data change a longstanding paradigm that PML is the central target of tumor mutations and viruses to now include the convergence on the TRIM family.
Introduction
Viruses and their hosts have co-evolved in the struggle for survival, both attempting to usurp the defensive strategies of the other. The host organism must acquire mechanisms to detect and destroy the foreign invader without destroying himself, whilst viral proteins mimic cellular structures to pirate the cell’s natural machinery for use in its own replication. This presents an obstacle for the host organism’s innate immune system, the first line of defense against viral replication and spread. One defining feature of many viral proteins is their ability to form oligomers. Less than 3% of human proteins form homo-oligomers (>trimer) whereas roughly half of early Adenoviral proteins do (unpublished data). Recently, the Tripartite Motif
(TRIM) superfamily of proteins has been revealed as having a role in innate immunity where numerous TRIMs have been shown to restrict viral replication [61, 231] and are regulated by
Interferons [62]. Most striking about this family is that virtually every domain within TRIM proteins can serve as a site for protein-protein interactions driving homo- and hetero-oligomerization within the TRIM family [74]. However, the significance of TRIM-TRIM interactions amongst the family is unclear.
The TRIM family consists of over 70 human members with broad functionality. The defining feature of the TRIM family is the conserved spacing of the domains at the N-terminus, composed of a RING finger, one or two B-box motifs and a coiled-coil (RBCC domain). Their C- termini vary among the family providing them with diverse functions and classify the TRIMs into eleven sub-families [62]. Many C-terminal domains also have oligomeric properties, and combined with the RBCC domains allow the TRIMs to form an extensive interactome.
Furthermore, the RBCC domain structure has evolved as a unique block early in metazoan evolution, often encoded by a single exon, suggesting that the ability of TRIMs to oligomerize was critical for human evolution [100]. Several TRIMs interact directly with viral proteins to restrict viral replication such as TRIM5 and TRIM15 with MLV and HIV-1 oligomeric polyprotein Gag [61, 74
232]. The oligomeric properties of TRIMs and viral proteins may be the key to their interactions and of TRIM anti-viral function.
Adenovirus early protein E4-ORF3 is a small (13kDa) protein that oligomerizes to form a
remarkable network of cables that weaves through the nucleus [38]. E4-ORF3 has been shown
to selectively mislocalize three TRIM proteins, PML (TRIM19), TIF1α (TRIM24) and TIF1γ,
(TRIM33) as well as the MRE11/RAD50/NBS1 (MRN) DNA repair complex [41, 42, 214, 215,
233]. The ability of E4-ORF3 to mislocalize its targets relies on its capacity to oligomerize [38].
E4-ORF3 oligomerization creates avidity-driven interactions. However, these interactions will
also rely on the oligomerization of E4-ORF3’s targets. Thus far, only four out of seventy human
TRIM proteins have been tested for interactions with E4-ORF3. The diverse functions of the
TRIM family including regulation of transcription, protein degradation, post-translational
modifications and innate immune signaling make the TRIMs an irresistible group for viral
targeting. Conversely, the oligomerization of E4-ORF3 serves as a perfect platform for TRIMs to
sense this foreign invader and subsequently activate innate immune functions.
E4-ORF3 is one of many viral proteins known to target TRIM protein PML. PML
(Promyelocytic Leukemia protein) was first discovered as a fusion protein in patients with Acute
Promyelocytic Leukemia where, due to a chromosomal translocation, the N-terminus of PML is
fused with the C-terminus of the Retinoic Acid Receptor-α [45]. Because of the convergence on
PML by viral proteins and tumor mutations, the PML protein was thought to act as a critical
regulatory protein such as archetypal DNA viral targets Retinoblastoma protein (Rb) [234] and
tumor protein 53 (p53) [3]. On the contrary, PML null mice did not get Leukemia and have only a
slightly higher cancer phenotype in ageing animals [57]. Furthermore, PML has not been shown
to have a significant impact on Adenovirus and Herpes Simplex virus-1 (HSV-1) replication [235].
It is often overlooked that PML belongs to the TRIM super-family where it homo-oligomerizes and
can hetero-oligomerize with multiple other TRIMs. We hypothesized that PML is not the central
TRIM target of viral proteins, and instead propose that oligomeric viral proteins that target PML
are actually targeting the TRIM family network. 75
Here we show that the broad functionality of the TRIM family by plotting its first order interactome that include over 900 proteins. We also analysize of the set of proteins in this protein network and reveals the contribution of the TRIM interactome in the regulation of many processes including cell cycle, apoptosis, and stress responses as well as the TGF-β, TNF-α, p53 and nuclear receptor pathways. Using a high-throughput immunofluorescence screen, we reveal novel interactions of a subset of TRIM proteins with oligomeric DNA virus proteins E4-ORF3,
HSV-1 ICP0 and CMV IE2. Moreover, we show that E4-ORF3 and ICP0 do not target a single
TRIM domain, and instead exhibit interactions with multiple domains within TRIMs. Furthermore, we use super-resolution imaging to show the various interaction phenotypes of TRIMs with E4-
ORF3 and ICP0.
Results
The TRIM family of proteins forms a protein-protein network based on hetero- oligomerization among TRIMs
The homo- and hetero- oligomerization properties of the TRIM family are undoubtedly vital to their synergistic functions. To illustrate the network of TRIM proteins generated by TRIM-TRIM interactions, we used the BisoGenet plugin of Cytoscape (using the SysBiomics database populated by protein interaction data from Uniprot, DIP, BIND, HPRD, MINT, Intact and BioGrid) to plot the TRIM interactome (Figure 3.1). TRIM proteins are represented by boxes color-coded by their respective C-termini to visualize interactions within TRIM subfamilies. It is immediately obvious that TRIMs with PHD-BROMO C-terminal domains (TRIM24, TRIM28 and TRIM33) form an interaction group. The same is true for TRIMs containing a COS domain (microtubule binding domain) (TRIM55, TRIM54 and TRIM63). Conversely, NHL domain containing proteins (TRIM32,
TRIM2, TRIM3 and TRIM71) do not have any known interactions within this subfamily. About half of TRIM proteins contain a C-terminal SPRY domain, however, this subfamily of TRIMs do not form a distinct interaction group, however most SPRY domain TRIMs within the network do exhibit connections with at least one other SPRY domain TRIM protein. The TRIM interactome also reveals distinct TRIM hubs in TRIM8, with 13 interactions and TRIM27 with 11 interactions. 76
Considering that most interactions have been found through in vitro yeast two-hybrid interaction studies, it is likely that interactions among TRIM proteins are dependent on many factors including subcellular localization, concentration and post-translational modifications. Thirty-five
TRIMs have no reported interactions with other TRIMs, however we believe that the known TRIM interactions are extremely limited. We have co-expressed TRIMs with no known interactions, such as TRIM61 with other TRIMs, such as TRIM22 and shown that they co-localize in the nucleus of U2OS cells (Supp. Figure 3.1). Although co-localization doesn’t confirm a direct protein-protein interaction, co-localization of proteins highly suggests that they function together in a protein complex. We were able to show co-localization between TRIM14 and 22, TRIM22 and 24, TRIM22 and PML, TRIM22 and 15, TRIM22 and 61, TRIM61 and 15, and TRIM15 and 24
(Figure 3.1, black edges, Figure 3.2B and Supp. Figure 3.1). These data illustrate that the known interactions among TRIM proteins is likely to be extremely narrow from what is possible in reality.
TRIMs homo- and hetero- oligomerize through all of their domains
The extraordinary feature of TRIM proteins is their multiple protein interaction domains.
To test which domains are sufficient to homo- or hetero-oligomerize with TRIMs from various subfamilies, we constructed TRIM domain mutants of TRIM22 and TRIM14, both with SPRY C- termini, TRIM61 which only contains an RBCC domain, and TRIM24, known for its functions in transcriptional regulation with a PHD-BROMO domain at its C-terminus. For example, the full lengthTRIM22 protein has a RING domain, B-box2 domain, coiled-coil and a SPRY domain at the
C-terminus. We constructed mutants tagged with GFP at the N-terminus with the RING alone,
RING + B-box2, B-box2 alone, B-box2 + coiled-coil, coiled-coil alone, coiled-coil + C-terminus, and the C-terminus alone (illustrated in Figure 3.2A). We then co-expressed the TRIM22 77
Figure 3.1 The family of TRIM proteins form a network based on protein-protein interactions.
The BisoGenet plugin in Cytoscape was used to generate the TRIM network. TRIMs are color coded based on their C-terminus as specified in the “Key for Nodes”. The method used to determine TRIM-TRIM interactions is specified in the “Key for Edges”. 78
domain mutants with full length FLAG-tagged TRIM22, 14, and 15. Remarkably, we found that all
TRIM22 domains alone were able to co-localize with full length TRIM22, 14 and 15 proteins
(Figure 3.2B-F, Supp. Figure 3.1 and summarized using cartoons in Figure 3.2 G-I). Analogous
TRIM domain mutants were constructed for TRIM14 (with a B-box2, coiled-coil and PRY-SPRY domains) which has no known interactions with other TRIMs, and co-expressed with full-length
TRIM22, 14 and 15. We found that full-length TRIM14 was able to co-localize with all TRIM14 domain mutants, while TRIM22 and 15 co-localized with the B-box2 and C-terminus, but not the
Coiled-coil (Supp Fig.1, summarized in Fig 2J-L). TRIM61 does not have a C-terminal domain and has no known interactions with any proteins. The TRIM61 RING, B-box2 and coiled-coil domain mutants were all able to co-localize with full-length TRIM22 and 15 (Supp. Figure 3.1, summarized in Figure 3.2M-N). Lastly, we constructed domain mutants in the same manner for
TRIM24, a TRIM with a RING, B-box1, B-box2, coiled-coil, and C-terminal PHD-BROMO domains along with an undefined (UD) region between the coiled-coil and PHD-BROMO domains.
TRIM24 domain mutants were co-expressed with full-length TRIM33, 22 and 15. Surprisingly, every domain mutant of TRIM24 including the UD region was able to co-localize with full length
TRIM22 and TRIM15 whereas TRIM33 co-localized with all except for the UD region (Supp.
Figure 3.1, summarized in Figure 3.2 O-Q). Clearly, co-localization is not tantamount to direct protein interactions, however, it highly suggests that each TRIM domain is capable of facilitating protein-protein interactions among the family of TRIM proteins illustrating the complexity of the
TRIM interactome.
The TRIM first order interactome suggests functions in a vast array of critical cellular pathways
The functions of the family of TRIM proteins are undoubtedly linked through interactions leading to distinct protein complexes. To elucidate the major functions of the TRIM family as a whole, we first created an interactome of TRIM proteins and their first order interactors again using 79
Figure 3.2 TRIM proteins interact with each other via multiple domains.
A. Schematic of N-terminal GFP tagged TRIM22 domain mutants. B-F, GFP tagged TRIM22 domain mutants were expressed alone, or co-expressed with FLAG tagged full length TRIM22 or TRIM14, as indicated, in U2OS cells. Cells were fixed and stained 24hrs after transfection for FLAG (red) and DNA (blue). G.-H. Schematic representations of the results from B.-F. where grey boxes represent a full length FLAG-tagged TRIM protein, as indicated, and black lines connecting the grey box to each TRIM domain represents a positive co-localization between the full length TRIM protein and the indicated domain mutant. L.-Q. Schematic representations of co-localization results from FLAG tagged full length TRIM proteins (grey boxes) with domain mutants, as indicated. Black connecting lines between domain mutants with full length TRIMs indicate a positive co-localization and the absence of a black line indicates no co-localization between the full length TRIM and domain mutant. Immunoflurescence images of data is found in Supp. Figure 3.1 80
the BisoGenet plugin for Cytoscape (Figure 3.3A). The resulting interactome contained 917 nodes (proteins) and 12,637 edges (interactions). To reveal which TRIMs are acting as protein interaction hubs, we ordered the TRIMs based on the number of interactions within the interactome (also called “degree”). PML, TRIM28 and 27 have over 100 interactions each, while
TRIM63, 8, 55 and 21 all have 60 or more (Figure 3.3B and Supp. Table 1). Furthermore, we ordered all proteins within the interactome by degree of interactions (Figure 3.3C and Supp.
Table 1). UBC (Ubiquitin C) has 736 interactions which is expected as it is able to interact with thousands of proteins. SUMO2, p53, PML, APP (Amyloid beta precursor protein), TRIM28,
SUMO1, ESR1 (Estrogen receptor), HS90A (Heat Shock protein), HDAC1, EP300, CUL3, 14-3-
3z, and UBC9 (SUMO E1) are the proteins within the interactome with the highest degrees, each containing over 120 interactions with other proteins within the network. Proteins within the first order interactome of the TRIM family are involved in a plethora of cellular functions. To determine which biological processes were most represented within the interaction, we used the list 1st order TRIM interactors and analyzed them using the Genomatix Pathway System (GePS) and GeneRanker programs. This analysis revealed that the genes within the extended TRIM interactome are involved in critical regulatory processes such as transcription, protein ubiquitination and sumoylaiton, apoptosis, stress response, cell cycle, DNA damage response,
NF-κB, and interferon production, among others (Figure 3.3D and Supp Table 2). Furthermore, the analysis identified TGF-β, NF-κB, HDAC signaling, nuclear receptor, p53, Wnt, and the toll- like receptor pathways as the top canonical pathways significantly over-represented by the protein network of first order TRIM interactors (Figure 3.3E and Supp Table 3). These data are evidence for the role of the TRIM family in regulation of multiple cellular pathways critical for cell growth and survival.
81
Figure 3.3 The network of TRIMs and first order interacting proteins reveal their likely functions in a wide array of cellular processes and pathways.
A. The network of the family of TRIM proteins (purple) with first order interacting proteins (blue). B. List of TRIM proteins with more than 40 interactions (degree). C. List of proteins with more than 100 interactions within the network in (A). D. List of top gene ontology (GO) Biological processes significantly overrepresented by the protein network in (A). Full data shown in Supp. Table 2. E. Selection of top canonical signal transduction pathways significantly overrepresented by the protein network in (A). Full data shown in Supp. Table 3. 82
Adenovirus E4-ORF3 interacts with a large subset of the TRIM family determined by protein concentration and subcellular localization
DNA viruses encode early virus proteins inducing quiescent cells into S-phase and simultaneously blocking apoptotic and anti-viral signals. Considering the conserved domain structure of the TRIM family and their roles in cell cycle, apoptosis and interferon and nuclear receptor regulation, it is rational to hypothesize that the family is disrupted by early viral proteins.
In fact, Adenovirus E4-ORF3 is able to mislocalize three TRIM proteins, PML, TRIM24 and
TRIM33, but not TRIM28. However, we hypothesized that E4-ORF3 targets additional TRIM proteins by mislocalization. To test this, we used a cell based immunofluorescence screen in which FLAG tagged TRIMs were co-expressed with E4-ORF3 in U2OS cells, fixed and stained
24hrs after transfection, and imaged with a high-throughput microscope (Figure 3.4). Co- localization was determined by eye where the conditions for a positive co-localization were non- pyknotic nuclei and co-localization of E4-ORF3 and TRIM in over half of co-transfected cells in either the nucleus and/or cytoplasm. 49 TRIM proteins were used in the screen (Supp. Table 4).
Co-localization was chosen over Immunoprecipitation because E4-ORF3 is highly insoluble and would most likely result in false negatives. Furthermore, the E4-ORF3 nuclear structure is very distinct and is known to mislocalize its known TRIM targets onto E4-ORF3 cables.
Using this screen we were able to identify 9 additional TRIM proteins displaying interactions with E4-ORF3 by co-localization (Fig 5). PML, TRIM24 and 33 were all mislocalized by E4-ORF3 as expected (Figure 3.5A-C). The phenotype of TRIM61 resembles that of TRIM33 where expressed alone localizes nuclear diffuse, and when co-expressed with E4-ORF3 is completely mislocalized with E4-ORF3 (Figure 3.5D). TRIM8, 22 and 35 also co-localize with nuclear E4-ORF3 (Figure 3.5E-G). Even though E4-ORF3 is mostly nuclear, it co-localized in the cytoplasm with TRIM14, 18, 45 and 48 in cytoplasmic bodies/speckles near to the nucleus
(Figure 3.5H-J and L). While TRIM55 and 29 displayed co-localization with E4-ORF3 in cytoplasmic fiber structures (Figure 3.5K and M). Not surprisingly, the levels 83
Figure 3.4 Schematic of high throughput screening to determine targets of E4-ORF3.
A. TRIM genes were either purchased in or cloned into Gateway Entry vectors. Using the gatway LR clonase, the TRIM genes were cloned into a DEST vector with the FLAG tag sequence at the N-terminus of the TRIM genes. B. FLAG-TRIMs were either transfected alone or with an expression vector for E4-ORF3, both expressed from the CMV promoter. C. 24hrs after transfection, cells were fixed and stained for FLAG (green), E4-ORF3 (red) and DNA (blue). The ImageXpress high-throughput imager was used with a 20X objective taking 12 images per condition. D. Images were analyzed by eye to determine co-localization of TRIMs with E4-ORF3. 84
Figure 3.5 E4-ORF3 mislocalizes a subset of the TRIM family in the nucleus or cytoplasm.
N-terminally tagged FLAG TRIMs (as indicated) were expressed alone or co-expressed with E4-ORF3 in U2OS cells. After 24 hrs, cells were fixed and stained for FLAG (green), E4- ORF3 (red) and DNA (blue). A.-G. show TRIM proteins that co-localize with E4-ORF3 in the nucleus and H.-M. in the cytoplasm. E.-G. and K.-M. show close-ups of regions indicated by a grey box in the merged image showing FLAG-TRIM (green), E4-ORF3 (red) and a merge of green and red. 85
Figure 3.6 MYC tagged E4-ORF3 accumulates in the nucleus and cytoplasm revealing additional potential TRIM targets.
A. Comparison of untagged E4-ORF3 to MYC tagged E4-ORF3 expressed in U2OS cells after 24 hours B.-C. Comparison of TRIM35 or TRIM74 (as indicated) co-expressed with E4-ORF3 untagged vs MYC-E4-ORF3. D.-M. N-terminally tagged FLAG TRIMs (as indicated) were expressed alone or co-expressed with MYC-E4-ORF3 in U2OS cells. After 24 hrs, cells were fixed and stained for FLAG (green), E4-ORF3 (red) and DNA (blue). M. also shows a close-up region indicated by a yellow box in the merged image showing FLAG-TRIM63 (green), MYC- E4-ORF3 (red) and a merge of green and red. 86
of TRIM and E4-ORF3 expression were critical for their interactions. Therefore, we tested whether higher levels of E4-ORF3 protein, with more accumulation in the cytoplasm would reveal further TRIM-E4-ORF3 interactions.
Placing a MYC tag at the N-terminus of E4-ORF3 leads to thicker nuclear cables and more accumulation in the cytoplasm (Figure 3.6A). Co-localization between E4-ORF3 and TRIMs was much more conspicuous when MYC-E4-ORF3 was expressed in lieu of untagged E4-ORF3.
For example, TRIM35 co-localizes with untagged E4-ORF3 in small nuclear speckles, however when expressed with MYC-E4-ORF3, the co-localization in the nucleus and cytoplasm is unmistakable (Figure 3.6B). We re-screened the TRIM proteins for co-localization with MYC-E4-
ORF3 and revealed 11 additional TRIMs able to co-localize with MYC-E4-ORF3 (Figure 3.5 and
Supp. Fig 2). Of these, TRIM74 was the only TRIM protein distinctly mislocalized into nuclear cables with MYC-E4-ORF3 (Figure 3.6C). TRIM1 and 25 co-localize with MYC-E4-ORF3 in cytoplasmic fibers, while
TRIM15, 31, 32, 37, 39, 43, 50 and 63 co-localize in cytoplasmic bodies or speckles
(Figure 3.5D-M). In total, 20 TRIM proteins were able to co-localize with E4-ORF3 on top of its 3 known TRIM targets. Furthermore, we re-screened the family of TRIM proteins for co-localization with E4-ORF3 proteins from disparate subgroups. The aforementioned analyses were performed for E4-ORF3 from Ad5 of Subgroup C. MYC tagged E4-ORF3 proteins from Ad9 Subgroup D,
Ad12 Subgroup A and Ad34 Subgroup B were co-expressed with FLAG tagged TRIMs and analyzed using the same method as for Ad5 E4-ORF3. We found that all TRIM proteins that co- localized with Ad5 E4-ORF3 were also able to co-localize with at least one additional E4-ORF3 protein from a different subgroup except for TRIM32 (Supp. Figure 3.2). This reveals that the targeting of TRIM proteins is a conserved function of E4-ORF3 across disparate adenovirus serotypes. Adenoviruses from different subgroups possess varied tropisms infecting lung, kidney, and epithelial cells among others. TRIM interactions with E4-ORF3 proteins may only be relevant in certain cell types.
87
HSV-1 ICP0 mislocalizes and degrades a large subset of the TRIM family
E4-ORF3 is one of many viral proteins shown to target PML. HSV-1 ICP0 is an E3 Ub
Ligase that binds to PML during early infection and promotes degradation of PML. No other
TRIM proteins have been reported to interact with ICP0 however, we hypothesized that, like E4-
ORF3, ICP0 has a broader function in targeting the TRIM family, and not only PML. To test this, we used a similar approach as used with E4-ORF3 where ICP0 was co-expressed with FLAG- tagged TRIMs in U2OS cells and imaged to look for co-localization. ICP0, however, degrades
PML and in this assay would not co-localize. Therefore, we also treated cells with the proteosome inhibitor, MG-132 and co-transfected TRIMs with a mutant of ICP0 lacking the RING domain (∆RING-ICP0) given that ICP0 may be able to degrade additional TRIMs. Using this method, we identified 11 TRIMs that exhibited strong co-localization patterns with wild-type ICP0 even in the absence of MG-132. TRIM8, 10, 22, 35, 37, 38, 48, 51, 52, 61 and 74 co-localize with
ICP0 in characteristic nuclear ring shaped bodies (Figure 3.7A-I). TRIM8, 22 and 35 expressed without ICP0 were already located in discrete nuclear bodies, however TRIM10, 37 expressed alone are mostly cytoplasmic diffuse, but are mislocalized into the nucleus within ICP0 ring structures (Figure 3.7A-E). TRIM 38 alone localizes into fibrous structures within the nucleus, and around the nuclear periphery and is mislocalized into nuclear bodies co-localizing with ICP0
(Figure 3.7F). Lastly, TRIM48, 51, 52, 61 and 74 alone have nuclear diffuse localization that is mislocalized into ICP0 rings. The stabilized co-localization between these TRIMs and wild-type
ICP0 suggests that these TRIMs are not targeted for ubiquitination by ICP0. Alternatively, we found that TRIM24, 27, 26, 41, 33, 15 and 45 all display signs of degradation by ICP0 as they either have minimal or no co-localization with ICP0 wild-type, but do with the addition of MG-132 or expression with ∆RING-ICP0 (Figure 3.8). ∆RING-ICP0 displays a similar subcellular localization to wild-type ICP0 with slightly higher accumulation in the cytoplasm of transfected cells (Figure 3.8A). In our assay for co-localization, endogenous PML was eliminated by wild- type ICP0, while MG-132 addition restored co-localization of PML and ICP0, and expression of
∆RING-ICP0 mislocalized PML into characteristic ICP0 nuclear rings which validated our co- 88
localization assay for identifying TRIMs capable of being degraded by ICP0 (Figure 3.8B).
TRIM24, 27, 26, 41 and 45 do not co-localize with wild-type ICP0 in the absence of MG-132, however, upon addition, or with co-expression of ∆RING-ICP0, each TRIM is mislocalized into nuclear ring structures (Figure 3.8C-F and I). TRIM33 and 15 displayed minimal co-localization with wild-type ICP0, but significant co-localization with ∆RING-ICP0 either in nuclear ring
structures, or cytoplasmic bodies, respectively (Figure 3.8G-H). To confirm the results of our
assertion that some TRIM proteins are degraded by ICP0 and others are not, we infected U2OS
cells with wild-type HSV-1 or HSV-1 in which ICP0 is deleted (∆ICP0). We also added MG-132 to
infected cells to confirm proteasome dependent degradation. After 6 hours of infection, cells
were harvested and lysates subjected to western blot analysis using endogenous antibodies for
well known TRIMs with established working antibodies. We show that TRIM33 and 27 proteins
are degraded during wild-type HSV infection, and that TRIM22 is not, as expected from our co-
localization results. This degradation was dependent on ICP0 and the proteosome as ∆ICP0 and
the addition of MG-132 to wild-type HSV-1 prevented degradation of TRIM33 and 27 (Figure
3.8J). These results confirm that TRIMs that co-localize with ICP0 only with MG-132 or the
∆RING-ICP0 mutant, are in fact degraded due to ICP0 expression.
A subset of 12 TRIMs is converged upon by E4-ORF3 and ICP0
To compare the two sets of TRIMs that interact with E4-ORF3 or ICP0, we created a
Venn diagram illustrating the sets of TRIMs that co-localize with E4-ORF3, ICP0, or both (Figure
3.9). It is notable that of these subsets of TRIMs, there is not one domain that is conserved
among all targeted TRIMs. Therefore, either E4-ORF3 and ICP0 interact with multiple TRIM
domains, or some TRIMs are interacting via additional binding partners. The Venn diagram
reveals that 12 TRIMs have interactions with E4-ORF3 and ICP0, namely TRIM8, 15, 22, 24, 33,
35, 37, 45, 48, 61, 74 and PML. Interestingly, TRIM8, 35 and 74 are able to interact with each
other, while TRIM22, 61, 15 interact and TRIM24 and 33 also have an interaction (Figure 3.1).
Furthermore, TRIM22 interacts with TRIM24 and PML while 89
Figure 3.7 ICP0 mislocalizes a subset of the TRIM family.
N-terminally tagged FLAG TRIMs (as indicated) were expressed alone or co-expressed with ICP0 in U2OS cells. After 24 hrs, cells were fixed and stained for FLAG (green), ICP0 (red) and DNA (blue). B. also shows a close-up region indicated by a gey box in the merged image showing FLAG-TRIM63 (green), ICP0 (red) and a merge of green and red. 90
Figure 3.8 ICP0 causes degradation of a subset of TRIM proteins.
A. Comparison of subcellular localization of ICP0 and ∆RING-ICP0 expressed in U2OS cells after 24 hours B.-I. N-terminally tagged FLAG TRIMs (as indicated) were expressed alone or co-expressed with ICP0, ICP0 with proteosome inhibitor MG-132 added 4hrs before fixation, or ∆RING-ICP0 in U2OS cells (as indicated). 24hrs after transfection, cells were fixed and stained for FLAG (green), ICP0 (red) and DNA (blue). J. U2OS cells were infected with HSV-1 wild-type (wt) or HSV-1 with the ICP0 gene deleted (HSV ∆ICP0). MG-132 was added to cells at the time of infection. 6 hours post infection (hpi) cells were harvested for lysates and used in a western blot probed for ICP0, endogenous TRIM33, TRIM27 and TRIM22. 91
Figure 3.9 Venn diagram of TRIMs revealed to interact with E4-ORF3 and ICP0.
TRIM proteins co-localizing with E4-ORF3 or ICP0 are shown with their domain structures defined by the boxed key below the Venn diagram. TRIM proteins with a * are TRIMs mislocalized by MYC-E4-ORF3 and not untagged E4-ORF3. 92
TRIM15 interacts with TRIM8. These data illustrate that interactions among the TRIM proteins are likely to facilitate interactions with viral proteins E4-ORF3 and ICP0. Another notable observation is that 9 out of 12 TRIMs that interact with E4-ORF3 and not ICP0 only co-localize with MYC-E4-ORF3 and not untagged E4-ORF3 (Figure 3.1 starred TRIMs). This suggests that
E4-ORF3 accumulation to high levels in the nucleus and cytoplasm drive interactions among additional TRIM interacting groups such as TRIM32, 1, 18 and 29 or TRIM55 and 63 (Figure 3.1).
E4-ORF3 and ICP0 do not converge in targeting a common TRIM domain and instead are able to mislocalize multiple TRIM domains
To test if the majority of TRIMs that co-localize with E4-ORF3 and ICP0 TRIMs are directly targeted via a common TRIM domain, we co-expressed E4-ORF3 and ∆RING-ICP0 with
GFP tagged TRIM domain mutants of common targets TRIM22, 24 and 61, and E4-ORF3 target
TRIM14, constructed as described earlier (Figure 3.2A). We chose TRIM22 and 14 because they have C-terminal SPRY domains found in over half of the TRIM family, TRIM61 because it is only composed of the RBCC region with no C-terminus and TRIM24 because it has been shown to be a target of E4-ORF3 in previous studies. We found that surprisingly, TRIM22 and 14 C-terminal domains, each containing a SPRY domain, were sufficient for co-localization with E4-ORF3, while all other TRIM22 and 14 domains were not (Figure 3.10A-E, summarized in F and L with additional data in Supp. Figure 3.1); while, TRIM24 and 61 coiled-coiled domains alone were sufficient for co-localization with E4-ORF3 (Figure 3.10G-J, M-P, summarized in K and Q. with extended data in Supp. Fig 1). The coiled-coil domain of TRIM24 was reported in previous studies to be sufficient for an interaction with E4-ORF3 [41], however, we report that the undefined region between the coiled-coil and the PHD-BROMO domain are each sufficient for mislocalization by E4-ORF3.
The same analysis assaying co-localization of E4-ORF3 and GFP-TRIM domain mutants was used to test interactions between ICP0 and TRIM22, 24 and 61 domain mutants, however
93
Figure 3.10 E4-ORF3 does not target a single TRIM domain.
A.-E., G.-J., M.-Q. GFP tagged TRIM22, TRIM61 or TRIM24 full length proteins or domain mutants (as indicated) were expressed alone, or co-expressed with E4-ORF3 in U2OS cells. Cells were fixed and stained 24hrs after transfection for E4-ORF3 (red) and DNA (blue). F. K. and L. Schematic representations of the interaction results between E4-ORF3 (grey box) and TRIM domain mutants (cartoon ). Black lines connecting the grey box to each TRIM domain represents a positive co-localization between E4-ORF3 and the indicated domain mutant while the absence of a black line indicates no co-localization between E4-ORF3 and the indicated TRIM domain mutant. Immunoflurescence images of all domain mutant data is found in Supp. Figure 3.1 94
domain mutants were co-expressed with ∆RING-ICP0 to minimize confusion due to protein degradation. We found that the RING and C-terminus with SPRY domains of TRIM22 were sufficient for co-localization with ∆RING-ICP0 (Figure 3.11A-F). Surprisingly, all domains of
TRIM61, the RING, B-box2 and coiled-coil domains were sufficient for co-localization with
∆RING-ICP0 in nuclear rings (Figure 3.11G-K). Finally, TRIM24 RING and coiled-coil domains
were sufficient for co-localization with ∆RING-ICP0, however, we also found that the undefined
region and PHD-BROMO domains were able to co-localize with ∆RING-ICP0, only when
expressed as a unit, and not when the domains were expressed alone with ∆RING-ICP0 (Figure
3.11L-P and Supp. Figure 3.1). Interestingly, when the undefined and PHD-BROMO domains are
expressed as a unit with E4-ORF3, they are unable to co-localize, but when each domain is
expressed alone with E4-ORF3, they are both capable of interacting (Figure 3.10 and Supp.
Figure 3.1). Taken together these data suggest that E4-ORF3 and ICP0 are capable of
interacting with multiple domains within TRIM proteins and do not target a single domain.
However, the caveat to the experiment is that there are endogenous TRIM proteins that may be
facilitating an interaction between TRIM domain mutants and the viral proteins.
Super-resolution microscopy reveals details of TRIM interactions with E4-ORF3 and ICP0.
Super-resolution microscopy is able to resolve subcellular structures by “breaking the diffraction limit of light” [236]. Structure illumination microscopy (SIM) uses patterned illumination relying on both specific microscopy protocols and extensive software analysis post-exposure.
While a normal wide-field microscope has a resolution of about ~250 nm, SIM technology can resolve structures down to ~100 nm. Seeing as the co-localization results of E4-ORF3 and ICP0 with TRIM proteins were imaged with a wide-field microscope, we found it useful to image the co- localization using super-resolution SIM technology. MYC-E4-ORF3 was co-expressed with either
FLAG-TRIM33 alone or with GFP-TRIM24 or GFP-TRIM61. Co-expression of two TRIMs simultaneously with E4-ORF3 allows us to witness the interactions among TRIMs at the same time as interactions between TRIMs and E4-ORF3. We predicted from earlier results 95
Figure 3.11 ICP0 mislocalizes TRIM proteins via multiple domains within each TRIM.
A.-E., G.-J., L.-O. GFP tagged TRIM22, TRIM61 or TRIM24 full length proteins or domain mutants (as indicated) were expressed alone, or co-expressed with ∆RING-ICP0 in U2OS cells. Cells were fixed and stained 24hrs after transfection for ICP0 (red) and DNA (blue). F. K. and P. Schematic representations of the interaction results between ICP0 (grey box) and TRIM domain mutants (cartoon ). Black lines connecting the grey box to each TRIM domain represents a positive co-localization between ∆RING-ICP0 and the indicated domain mutant while the absence of a black line indicates no co-localization between ∆RING-ICP0 and the indicated TRIM domain mutant. Immunoflurescence images of all domain mutant data is found in Supp. Figure 3.1 96
that TRIM proteins are likely to interact with each other and promote interactions with E4-ORF3 creating a layering effect. This is exactly what we see when FLAG-TRIM33 and GFP-TRIM24 are co-expressed with MYC-E4-ORF3 where TRIM33 is sandwiched between ORF3 and TRIM24 implying that TRIM24’s interaction with TRIM33 is facilitating its co-localization to E4-ORF3
cables (Figure 3.11A). Alternatively, when FLAG-TRIM33 is expressed alone with MYC-E4-
ORF3 and imaged with endogenous PML, we observe a very different pattern whereTRIM33 and
PML do not overlap and instead sit on top of E4-ORF3 cables adjacent to one another in a linear
chain with alternating domains of TRIM33 and PML (Figure 3.11B). Furthermore, the pattern of
localization for FLAG-TRIM33 co-expressed with GFP-TRIM61 and MYC-E4-ORF3, is again
vastly different where TRIM61 changes the E4-ORF3 cable structure and looks to be
incorporated within the E4-ORF3 matrix while E4-ORF3 fibers with integrated TRIM61 exclude
TRIM33 (Figure 3.11C). Taken together, we conclude that E4-ORF3 is directly binding to some
TRIM proteins and may use them to recruit additional TRIMs onto E4-ORF3 cables, while the E4-
ORF3 polymer has the capacity to incorporate proteins within its matrix which is likely to be a
mechanism by which it functions in viral replication.
Similar experiments were performed using SIM technology to resolve ICP0 nuclear ring
structures with TRIMs found to co-localize through the wide-field microscopy screen. ∆RING-
ICP0 was co-expressed with FLAG-TRIM22 alone and stained with antibodies to endogenous
PML or endogenous TRIM27, or co-expressed with GFP-TRIM61. SIM imaging reveals that the
ICP0 ring structure is for the most part hollow and that endogenous PML co-localizes directly onto
the edges of the ICP0 nuclear ring structure (Fig 12A). TRIM22, however, localizes to the inside
of the ICP0 ring creating an additional hollow ring within the ICP0 ring (Fig 12A and B). Like
PML, endogenous TRIM27 localizes directly on top of the ICP0 nuclear ring while again, TRIM22
forms a ring within (Figure 3.11B). TRIM61 has a completely different phenotype where it is
localized within the TRIM22 ring, inside of the ICP0 ring sandwiching TRIM22 in between TRIM61
and ICP0 (Figure 3.11C). Interestingly, PML and TRIM27 that localize on top of ICP0 rings are 97
Figure 3.12 Super-resolution images of TRIMs with E4-ORF3 reveals details of TRIM-E4- ORF3 interactions.
MYC-ORF3 was co-expressed with FLAG-TRIM33 and GFP-TRIM24 in A., FLAG-TRIM33 alone in B., or FLAG-TRIM33 and GFP-TRIM61 in C. 24hrs after transfection cells were fixed and stained for ORF3 (blue), FLAG (red), and in C. PML (green). DNA periphery is designated with a white line. Large images to the left are merged images with blue, red and green. Large images to the right are merged images showing red and green only. Center images represent close-ups of areas boxed with grey. 98
both targets of degradation by ICP0, while TRIM22 and TRIM61 are not (Figure 3.11, 8 and 9).
Taken together, these results reveal an extremely complex interaction profile for both E4-ORF3 and ICP0 with multiple members of the TRIM family. These results also suggest that homo- and hetero- oligomerization of TRIM proteins is used as a means for E4-ORF3 and ICP0 to mislocalize TRIMs. While it is still unclear which TRIMs interact directly with E4-ORF3 and ICP0, what we observe is that the mislocalization of TRIMs is occurring upon expression of these viral proteins, and that the mislocalization will undoubtedly affect the functions of the TRIMs.
Cytomegalovirus IE2 interacts with 7 TRIM proteins
We have shown that adenovirus E4-ORF3 and HSV-1 ICP0 target a large subset of the
TRIM family, however, the first TRIM discovered as a target of each, is PML. Cytomegalovirus
(CMV) also encodes proteins which disrupt PML bodies. IE1 drives de-sumoylation of PML, disrupting bodies early in CMV infection while IE2 does not disrupt PML, but accumulates at the periphery of PML bodies where it establishes viral DNA replication compartments [237-239]. We hypothesized that in addition to PML targeting proteins E4-ORF3 and ICP0, that IE1 and IE2 may also target additional TRIMs. To test this, we co-expressed HA tagged IE1 or IE2 with the 49
FLAG tagged TRIMs constructed for this study and screened for co-localization using high- throughput imaging as outlined earlier. We were unable to observe any co-localization of IE1 with any TRIM protein (data not shown). Alternatively, we found that IE2 was able to co-localize with
7 TRIM proteins. TRIM15, 27 and 22 form nuclear and cytoplasmic bodies when expressed alone, and are not mislocalized by IE2, but do co-localize in nuclear bodies (Figure 3.12A-C).
TRIM24 is nuclear diffuse when expressed alone, but is mislocalized into punctuate nuclear bodies co-localizing with IE2 (Figure 3.12D). TRIM33 and 61 are also nuclear diffuse when expressed alone, but with IE2 form large concentrated nuclear domains reminiscent of inclusion bodies (Figure 3.12E-F). Finally, TRIM51 expressed alone is diffuse in the nucleus and cytoplasm, while co-expression with IE2 induces a nuclear localization reminiscent of heterohromatin, which co-localizes with IE2. Nothing is known about TRIM51, which is not a 99
Figure 3.13 Super-resolution images of TRIMs with ∆RING-ICP0 reveals details of TRIM- ICP0 interactions.
∆RING-ICP0 was co-expressed with FLAG-TRIM22 alone in A. and B. or FLAG-TRIM22 and GFP-TRIM61 in C. 24hrs after transfection cells were fixed and stained for ICP0 (blue), FLAG (red), and in A. PML (green) or in B. TRIM27 (green). DNA periphery is designated with a white line. Large images to the left are merged images with blue, red and green. Large images to the right are merged images showing red and green only. Center images represent close-ups of areas boxed with grey. 100
classic TRIM, for it lacks a RING and B-box domain, but does have a coiled-coil and SPRY domain. The varied localizations of TRIMs with IE2 suggest diverse roles in the regulation of IE2.
E4-ORF3, ICP0 and IE2 associate with a common subset of TRIM proteins
To illustrate and compare TRIM interactors of disparate viral proteins E4-ORF3, ICP0 and IE2, we generated a Venn diagram (Figure 3.13). Six TRIM proteins, TRIM15, 22, 24, 33, 61 and
PML all have interactions with E4-ORF3, IE2 and ICP0. These TRIM proteins also share interactions with one another (Figure 3.1) suggesting a role for hetero-oligomerization in advancing interactions between the TRIMs and viral proteins. TRIM8, 35, 37, 45, 48 and 74 interact with both E4-ORF3 and ICP0 (Figure 3.13). TRIM8, 74 and 35 are known to interact as well, further supporting a role of TRIM hetero-oligomerization in TRIM-viral protein interactions.
TRIM27 is a major hub for cellular protein interactions and co-localizes with IE2, and is degraded by ICP0. TRIM51 also interacts with both IE2 and ICP0, however its functions are unknown. In conclusion, we have shown that 31 out of 49 TRIMs tested have interactions with either E4-
ORF3, ICP0 or IE2. This represents a very large portion of the TRIM family that is disrupted by early DNA virus proteins. The implications of TRIM family disruption are evident by their interactome of first order interacting proteins with roles from regulation of cell cycle and apoptosis, to interferon and nuclear receptor activation.
Discussion
Here we have shown that the super family of Tripartite Motif proteins collectively functions in major pathways critical for cellular replication, death, and immunity which are vital for human life. With their many protein-protein interaction domains, the TRIMs are a protein network that work together in complexes with countless combinations. Just as antibodies have a variable region that can essentially adapt to respond to novel pathogens, TRIMs may have similar abilities in innate immunity triggering complex forming upon viral infection to inhibit viral infection or alert 101
Figure 3.14 IE2 interacts with a subset of the TRIM family in diverse nuclear structures.
N-terminally tagged FLAG TRIMs (as indicated) were expressed alone or co-expressed with HA tagged IE2 in U2OS cells. After 24 hrs, cells were fixed and stained for FLAG (green), HA (red) and DNA (blue). B. and G. also show close-ups of regions indicated by a gey box in the merged image. Close-up features green alone (green), red alone (middle) and green/red overlay (right). 102
Figure 3.15 Venn diagram of TRIMs revealed to interact with E4-ORF3, ICP0 and IE2.
TRIM proteins co-localizing with E4-ORF3, ICP0 or IE2 are shown with their domain structures defined by the boxed key below the Venn diagram. 103
other cells to the invasion. We’ve revealed that disparate DNA virus proteins E4-ORF3, ICP0 and
IE2 all display interactions with multiple members of the TRIM family through several TRIM domains. Whether the functions of these TRIMs are inhibited by the viral proteins, or if the viral proteins are recruited as an anti-viral mechanism is still unclear. However, ICP0 is able to degrade at least 8 TRIMs suggesting that these are true targets of ICP0 in HSV-1 infection.
ICP0, like many of the TRIMs is an E3 Ubiquitin ligase with a RING domain and with at least 2 homo-oligomerization domains. Furthermore, ICP0 contains sumo interacting domains (SIMs) and uses them to stabilize interactions with PML [240]. Recently, TRIMs have been identified as
E3 SUMO ligases [241] and may also be sumoylated similarly to PML. It has been shown that sumoylation of the PHD-BROMO domain of TRIM28 changes the conformation of the domain altering the accessibility of TRIM28 domains [82]. Additional TRIM proteins may have similar properties when post-translationally modified revealing access to certain oligomeric domains depending on the cellular environment. It is also possible that ICP0 interacts with specific TRIMs to recruit in other TRIMs and may be using the E3 Ub ligase properties of those TRIMs to target others for degradation. Super-resolution images of TRIMs expressed with ∆RING-ICP0 revealed that different TRIMs had varied localizations within the ICP0 nuclear ring structure. We show that
PML and TRIM27 localize exclusively on top of ICP0 ring structures, while TRIM22 sits just inside of the ring, and TRIM61 localizes to the inside of the rings. Intriguingly, PML and TRIM27 are degraded by ICP0 whereas TRIM22 and TRIM61 are not.
E4-ORF3 higher order oligomerization creates a surface whereby cellular proteins are recruited through avidity-driven interactions [38]. The assembly of the E4-ORF3 polymer substantially reduces the ‘off-rate’ of low affinity interactions between individual E4-ORF3 dimer units and cellular partners by using multiple interaction points. Avidity-driven interactions require both partners to have multivalent binding sites [242]. TRIM proteins fulfill the requirement of multivalent binding sites providing evidence that the oligomerization of TRIMs is required for mislocalization by E4-ORF3. Because E4-ORF3 interactions with TRIMs are likely avidity-driven, the concentration and localization of each is critical for an interaction to occur. We show that 104
increasing the cytoplasmic concentration of E4-ORF3 reveals additional TRIM interactions.
Likewise, sites with high concentrations of TRIM proteins are likely to drive interactions with E4-
ORF3, as is evident by the mislocalization of E4-ORF3 into cytoplasmic bodies formed by TRIMs.
Interferon upregulation of TRIM proteins increases the concentration of multiple TRIMs simultaneously which likely drives TRIM homo- and hetero-oligomerization facilitating low-affinity interactions and perhaps forming novel anti-viral TRIM-complexes to detect and inhibit virus entry or oligomeric viral proteins. Evidence for this is seen in the restriction of HIV and MLV by TRIM5,
TRIM15 and 22. Individually they have been shown to restrict HIV replication by inhibition of the coat polyprotein Gag [61, 102, 232], however the interactions among TRIM5, 15 and 22 and their involvement in restriction of HIV have not been studied.
Super-resolution microscopy of TRIM proteins interacting with E4-ORF3 revealed distinct patterns for localization on E4-ORF3 fibers. We predicted that TRIM-TRIM hetero- oligomerization facilitates the interactions between E4-ORF3 and TRIMs, which would result in a layering effect with a TRIM layer in between E4-ORF3 and an additional TRIM. In fact, this is what we see with TRIM24 and 33, where TRIM33 forms a surface between TRIM24 and E4-
ORF3. In contrast, TRIM33 and PML do not overlap and instead sit on top of E4-ORF3 cables adjacent to one another in a linear chain with alternating domains of TRIM33 and PML. TRIM61, however, changes the E4-ORF3 cable structure and looks to be incorporated within the E4-ORF3 matrix. Furthermore, E4-ORF3 fibers with integrated TRIM61 exclude TRIM33. These results indicate that E4-ORF3 is directly binding to some TRIM proteins and may use them to recruit additional TRIMs onto E4-ORF3 cables. Furthermore, it illustrates that the E4-ORF3 polymer has the capacity to incorporate proteins within its matrix which is likely to be a mechanism by which it causes proteins to become insoluble.
One surprising result in this study was the mislocalization of CMV IE2 by multiple TRIM proteins. CMV IE1 drives desumoylation of PML and disruption of PML bodies, however we did not see any interactions among TRIMs and IE1. This co-localization assay was likely the wrong platform in which to study the effects of IE1 on TRIM proteins. However, we did reveal that IE2 105
co-localizes with several TRIMs in nuclear bodies and is highly concentrated into what look like inclusion bodies by TRIM33 and 61. One major difference between IE1 and IE2 is their ability to oligomerize. IE1 is not known to form homo-oligomers, whereas IE2 is known to dimerize and localizes to discrete nuclear bodies producing an area with high concentration of IE2 [243]. The formation of highly concentrated bodies when co-expressed with TRIM33 or 61 suggests that these TRIMs interact with IE2 and drive higher order oligomerization. It is compelling to suggest that the inclusion bodies are created by TRIM proteins to survive cell death and alert the adaptive immune response of the pathogen. Moreover, E4-ORF3 and ICP0 are insoluble and may also survive cell death, coated with TRIM proteins.
The role of the TRIM family in anti-viral activities of DNA viruses is still unclear.
Unpublished studies show that TRIM over-expression in U2OS cells does not inhibit first round replication of Adenovirus or HSV-1. However, TRIM33 over-expression in HeLa cells was able to abrogate Adenovirus replication, while its knock-down facilitated replication [214]. Therefore, the cell type and conditions under which TRIMs are over-expressed are critical for their anti-viral functions. Furthermore, the number of TRIMs has rapidly expanded very recently in evolution of higher eukaryotes [64, 100] suggesting that new TRIMs were needed for human survival against viral pathogens. This also suggests that current viral pathogens have evolved to subvert the activities of TRIM family members, as these viruses are still being spread amongst the human population. Therefore, the interactions revealed in this work between viral proteins and TRIMs are products of the co-evolution of humans and viruses in the endless battle for survival.
Materials & Methods
Cells
Human bone osteosarcoma cell line U2OS cells were cultured in Dulbecco modified
Eagle medium (DMEM, Invitrogen) supplemented with 10% (vol/vol) Fetal Calf Serum (FCS), kept at 37°C, in a humidified 5% CO2 atmosphere.
Plasmids and transfection 106
TRIMs were purchased in Gateway entry clones, or cloned into Gateway vector pENTR
(Invitrogen). A list of TRIM accession numbers used in this study is in Supp. Table4. Via
Gateway LR recombination [228], the TRIM genes were fused with the FLAG sequence in frame at the N-terminus with a DEST vector pcDNA3 backbone and expressed via CMV promoter.
TRIM domain mutants were cloned into Gateway entry vectors and using gateway LR recombination, the domain mutants were fused with the GFP sequence in frame at the N- terminus using pDEST53 (Invitrogen) and expressed via CMV promoter. Plasmids were transfected into U2OS cells using Lipofectamine 2000 per user instructions.
TRIM Network analysis
The BisoGenet plugin was used in conjuction with the cytoscape program to generate the
TRIM only network, and the network of TRIM plus first order interactors. The complete family of
TRIM proteins was used as inputs for both networks. Degrees of protein interactions were determined from the BisoGenet plugin. The genomatix GePS software was used to analyze the list of first order TRIM interactors which calculates p-values based on the number of genes corresponding to either the list of genes involved in specific Gene Ontology terms, or Canonical
Pathways.
Western Blots
Cells were lysed using RIPA buffer (100mM Tris pH 7.4, 300mM NaCl, 2mM EDTA, 2%
Triton X-100, 2% Deoxycholic acid, 2mM NaF, 0.2mM NaVO4, 2mM DTT, 0.1% SDS) with mini- protease cocktail (Roche), 10mM NEM, 0.5mM PMSF, 10nM Calyculin A, 5uM TSA, 5mM
Sodium butyrate, and three rounds of 30s sonication with amplitude of 65. 50ug protein lysate was loaded per well in pre-cast 4-20% Tris-Glycine gels (Invitrogen). Proteins were transferred using the Bio-Rad wet transfer system. Blots were blocked with 5% powdered milk in TNT (0.5M
TrisCl pH 8.0, 1.5M NaCl, 1% Tween20). Primary antibodies used for western blots: TRIM27
(Cell Sig), TRIM33 (Bethyl), ICP0 (Santa Cruz Biotech), TRIM22 (GeneAtlas). Primary antibodies were detected with secondary antibodies labeled with either IRDye800 (Rockland), or Alexa Fluor 107
680 (Molecular Probes). Fluorescent antibodies were visualized using a LI-COR-Odyssey scanner.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 30min, permeabilized with .1% Triton X-
100 for 5min and blocked with 5% Normal Goat Serum (Jackson ImmunoResearch) in PBS for
20mins. Cells were incubated with primary antibodies for 1 hour and washed once for 1min and twice for 20min. Cells were then incubated with secondary antibodies for 1 hour and washed once for 1min and twice for 20min (Alexa Flour 488, 555, and 633 from Invitrogen). Slides or 96- well plates were then rinsed with diH2O and stained with Hoechst. 96-well plates were unmounted and imaged immediately after staining with PBS covering cells. Slides were mounted with ProLong Gold (Invitrogen). High-throughput images were taken with a 40X ELWD objective using the ImageXpress and MetaXpress software. Repeats of insufficient images obtained from the ImageXpress were acquired using a Zeiss AxioPlan 2 microscope at 63X using AxioVision 4.6 software. Structured Illumination Microscopy was performed using a Zeiss Elyra PS.1 Super-
Resolution Microscope. Images were edited using programs ImageJ [230] and Photoshop
(Adobe). Primary antibodies used for IF: PML (PG-M3, Santa Cruz Biotech), TRIM33 (Abcam),
FLAG-tag (Sigma), Ad5 E4-ORF3 (Ascenion), ICP0 (Santa Cruz Biotech), HA tag (Sigma).
Author Contributions Kristen Espantman performed all experiments in Chapter 3. I thank Dr. Clodagh O’Shea for being a driving force behind the project in Chapter 3 and supporting my conviction in using the entire family of TRIM proteins throughout this work. I also thank Erik Kapernick for his valuable help using the ImageXpress microscope and software.
108
Conclusions and Closing Remarks
The similarities between DNA virus infected cells and cancer cells is striking. This is due
to the overlap in the targets of viral proteins and gene mutations that drive the cell into a
replicative state while blocking apoptosis and immune signaling. Not only has this overlap lead to
the discovery of critical cellular proteins that regulate cell growth and survival, but it has also
allowed us to develop oncolytic viral therapies using properties of cancer cells to compensate for
the deletion of key viral proteins. The ideal oncolytic virus would be able to treat many types of
cancer while being extremely selective for high levels of replication in cancer cells with no
replication in normal cells. Seeing as the p53 pathway is disrupted in the majority of cancers, the
loss of p53 activity is a desirable target for an oncolytic virus. Typically, p53 is targeted by viral
proteins by direct binding and subsequent inhibition, as is the case for Adenovirus E1B-55K. For
over 20 years it was believed that E1B-55K was the sole mechanism by which p53 was
inactivated in adenovirus infection. However, we discovered that Adenovirus E4-ORF3 prevents
the activation of p53 target genes using an unprecedented mechanism by inducing
heterochromatin formation specifically at genes regulated by p53. Only when E1B-55K and E4-
ORF3 are deleted are p53 target genes activated. p53 activation in normal cells by an oncolytic
adenovirus meant to selectively replicate based on p53 status is absolutely necessary.
Therefore, it was essential to reveal that E4-ORF3 also inactivates the p53 pathway in adenovirus
infection for the design of a p53 selective oncolytic adenovirus. Unfortunately, an adenovirus deleted for both E1B-55K and E4-ORF3 does not replicate, even in the absence of p53 activity.
Hence, it was imperative to find if it was possible to separate E4-ORF3’s essential functions in viral replication from its ability to inactivate p53 target genes.
E4-ORF3 proteins from disparate subgroups of adenovirus have limited amino acid sequence conservation, and in fact, E4-ORF3 from subgroup C is evolutionarily divergent from all other subgroups. E4-ORF3 from subgroup C is also where we discovered a function in inactivation for p53 target genes. If E4-ORF3 proteins from disparate subgroups were also able to block p53 activation, comparison of their conserved residues would point to the critical amino 109
acids for this function. Alternatively, if disparate E4-ORF3 proteins were not able to inactivate p53 target genes, sequence comparison of the non-conserved residues would point to the amino acids responsible for its acquired function. We revealed that p53 target gene inactivation is specific to E4-ORF3 from subgroup C. Furthermore, we show that mislocalization of the MRN complex is also specific to E4-ORF3 subgroup C while mislocalization of known targets PML,
TRIM24 and TRIM33 is a function of all E4-ORF3 proteins. This discovery allowed us to conclude that mislocalization of PML, TRIM24 and 33 were not sufficient for p53 target gene inactivation, and that there are natural mutations among E4-ORF3 proteins that separate E4-
ORF3’s functions. Comparison of the E4-ORF3 protein sequences from subgroups A, B, C and
D provided insight into which E4-ORF3 residues are necessary for p53 inactivation, versus PML mislocalization. This detailed information about E4-ORF3 will be used in the rational design of a p53 selective oncolytic adenovirus in which E4-ORF3 from subgroup C can be mutated as to retain its required functions in viral replication while revoking its ability to inactivate p53 target genes. Alternatively, we suggest that a p53 selective oncolytic virus be engineered from subgroup B adenoviruses in which E4-ORF3 does not inactivate p53 target genes, and for which there is low seroprevalence in the human population.
DNA virus proteins from disparate viruses often converge in targeting the same cellular proteins to initiate DNA replication and block death and anti-viral signals. Viral proteins also often function to deregulate multiple pathways at once to advance viral replication and block anti-viral signaling. Here we show that viral proteins from adenovirus, herpes simplex virus-1 (HSV-1) and cytomegalovirus (CMV) encode proteins originally known for targeting PML, actually target multiple members of the Tripartite Motif (TRIM) family of proteins, of which PML is a member.
Moreover, we suggest that a subset of TRIMs may have evolved to target viral proteins, by direct binding and inhibition, or possibly binding and downstream signaling to the innate or adaptive immune system. We show that through TRIM interactions with each other and additional cellular proteins, that the family of TRIMs has functions in regulating cell cycle, apoptosis, transcription regulation and regulation of major signaling pathways such as the TGF-β and NFκB pathways. 110
These data suggest that adenovirus E4-ORF3 and HSV-1 ICP0 target the TRIM family, and not
only PML, to deregulate multiple pathways simultaneously. Furthermore, we show that the
hetero-oligomerization of the TRIMs likely contributes to the interactions between TRIMs and viral
proteins, and that their localization with respect to the viral proteins determines whether they are the targets of, or are targeting the viral proteins. CMV IE2 does not disrupt PML bodies, but instead initiates viral replication centers at the periphery of PML bodies. The association of IE2 and TRIM proteins may reveal them as targets of IE2, or more likely, that TRIMs have evolved to detect the concentration of a foreign protein near PML bodies, as multiple viral proteins are known to localize at PML bodies. Taken together, these data reveal a complex interaction profile among the TRIM family, their cellular binding proteins, and viral proteins. The revelation that
TRIM proteins, an interconnected family, are targets of multiple viral proteins will hopefully lead to studies observing the effects of multiple TRIM proteins at once, instead of the isolated effects of one. For example, TRIM5, 15 and 22 restrict HIV or MLV replication through interactions with the
Gag polyprotein, however these TRIMs have not been tested for interactions with each other and if they work together to restrict viral replication. Furthermore, this work will change the longstanding paradigm that PML is the central target of viral proteins to now include convergence on the network of TRIMs.
APPENDIX
Chapter 2 Supplementary Tables
Supplementary Table 2.1 - List of Adenovirus protein IDs from NCBI used in this study.
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Supplementary Table 2.2 - Calculations of Surface Exposed Residues of E4-ORF3 as determined by the crystal structure of N82 E4-ORF3 from Ad5. GETAREA 1.0 beta was used to calculate the solvent accessible surface area of N82 E4-ORF3 (http://curie.utmb.edu/area_man.html). “Residue” represents an amino acid within N82 E4-ORF3 with residue position to the right of the residue name. “Total” represents the total solvent accessible surface area (backbone + side chain). “Backbone” represents the solvent accessible area contributed by the amino acid backbone. “Sidechain” represents the solvent accessible area contributed by the amino acid side chain. “Ratio” represents the ratio of sidechain solvent accessible area to surface area of “random coil” value of the same residue, expressed in a percentage. The "random coil" value of a residue X is the average solvent-accessible surface area of X in the tripeptide Gly-X-Gly in an ensemble of 30 random conformations. Residues are considered to be solvent exposed if the ratio value exceeds 50% and to be buried if the ratio is less than 20%, marked as "o" and "i" respectively in the last column labeled “In/Out”. Blank spaces in the In/Out column are residues whose Ratio % are between 20-50%. For this paper we used a cutoff of 40% where residues with ratios above 40% are considered solvent exposed and under 40% are not.
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Chapter 3 Supplementary Figures
Supplementary Figure 3.1 – Interaction Results of TRIM domain mutants co-expressed with full length TRIMs or viral proteins. GFP tagged full length TRIMs or TRIM domain mutants (as indicated at top and by cartoon) were expressed alone or with E4-ORF3, MYC- E4-ORF3 (E4-ORF3-myc), ICP0, ∆RING-ICP0 , or full length FLAG-TRIMs (as indicated to the
left of each image). Close-up images are provided where necessary designated by yellow boxes in merged image. Close-ups show green alone, red alone and green/red overlay. Cells were fixed 24 hrs after transfection and stained for E4-ORF3, FLAG or ICP0 (red) and DNA (blue). “Co-localization” denotes a positive observation of co-localization between the viral protein or FLAG tagged TRIM protein labeled to the left, and the GFP tagged TRIM domain
mutant labeled at the top while “No co-localization” denotes a negative observation of co- localization.
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Supplementary Figure 3.2 – Interaction Results of FLAG-TRIMs co-expressed with disparate viral proteins. FLAG-TRIMs were expressed alone or with ICP0, ∆RING-ICP0 (ICP0-FXE), Ad5 E4-ORF3 (E4-ORF3), Ad5 MYC-E4-ORF3 (E4-ORF3-myc Ad5), Ad34 MYC- E4-ORF3 (E4-ORF3-myc Ad34), Ad12 MYC-E4-ORF3 (E4-ORF3-myc-Ad12), Ad9 MYC-E4- ORF3 (E4-ORF3-myc Ad9), or HA-IE2 (IE2-HA) (as indicated to the left of each image). Close-up images are provided where necessary designated by yellow boxes in merged image. Close-ups show green alone, red alone and green/red overlay. Cells were fixed 24 hrs after transfection and stained for E4-ORF3, ICP0 or HA (red) and DNA (blue). “Co-localization” to the right of each image designates a positive result for co-localization of the TRIM and viral protein and “No co-localization” designates a negative result. 148
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Chapter 3 Supplementary Tables
Supplementary Table 3.1 – Degrees of protein-protein interactions within the network of the TRIM family of proteins and their first order interactors. The TRIM family of proteins was used as Input Identifiers within Cytoscape using the BisoGenet plugin (using the SysBiomics database populated by protein interaction data from Uniprot, DIP, BIND, HPRD, MINT, Intact and BioGrid) to generate a protein-protein interaction network by adding neighbors of input proteins of a distance of 1 (first order interactors). The entire list of proteins generated using this method is listed under the “TRIMs + 1st order interactors: Uniprot ID” with the number of protein-protein interactions within the network listed as “Deg.”. Under the heading “TRIMs only: Protein” shows the degree “Deg.” Of interactions within the TRIM family.
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Supplementary Table 3.2 – Top Gene Ontology (GO) Biological Processes significantly overrepresented by the protein network of first order TRIM interacting proteins. The 853 proteins reported to interact with the entire family of Human TRIM proteins were analyzed using the Genomatix Pathway System (GePS) and GeneRanker programs. Overrepresentation of Gene Ontology categories within the input gene list were calculated and are listed together with their respective p-value. The ‘Total # Genes’ refers to the total number of genes in the GO category; ‘Genes Exp.’ refers to the number of genes that would be expected at random; ‘Genes Obs.’ Refers to the number of genes within the input list associated with the GO category.
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Supplementary Table 3.2 – continued
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Supplementary Table 3.2 – continued 203
Supplementary Table 3.2 – continued
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Supplementary Table 3.2 – continued
. 206
Supplementary Table 3.3 – Top canonical signaling transduction pathways significantly overrepresented by the protein network of first order TRIM Interacting proteins. The 853 proteins reported to interact with the entire family of Human TRIM proteins were analyzed using the Genomatix Pathway System (GePS) and GeneRanker programs. Overrepresentation of canonical signal transduction pathways (from BioCarta and NCI-nature, http://pid.nci.nih.gov/) within the input gene list were calculated and are listed together with their respective p-value. The ‘Total # Genes’ refers to the total number of genes in the canonical pathway; ‘Genes Exp.’ Refers to the number of genes that would be expected at random, ‘Genes Obs.’ Refers to the number of genes within the input list associated with the pathway. (showing up to p-value #E-5)
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Supplementary Table 3.3 – continued
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Supplementary Table 3.3 – continued
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Supplementary Table 3.3 – continued
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Supplementary Table 3.3 – continued
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Supplementary Table 3.4 – Tripartite Motif proteins (TRIMs) used in this study
References
1. Gross, L., Pathogenic properties, and "vertical" transmission of the mouse leukemia agent. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine, 1951. 78(1): p. 342-8.
2. Howley, P.M. and D.M. Livingston, Small DNA tumor viruses: large contributors to biomedical sciences. Virology, 2009. 384(2): p. 256-9.
3. O'Shea, C.C., DNA tumor viruses -- the spies who lyse us. Curr Opin Genet Dev, 2005. 15(1): p. 18-26.
4. Ziff, E.B., Transcription and RNA processing by the DNA tumour viruses. Nature, 1980. 287(5782): p. 491-9.
5. Fitzgerald, M. and T. Shenk, The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell, 1981. 24(1): p. 251-60.
6. Kalderon, D., B.L. Roberts, W.D. Richardson, and A.E. Smith, A short amino acid sequence able to specify nuclear location. Cell, 1984. 39(3 Pt 2): p. 499-509.
7. Scheffner, M., J.M. Huibregtse, R.D. Vierstra, and P.M. Howley, The HPV-16 E6 and E6- AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell, 1993. 75(3): p. 495-505.
8. Scheffner, M., B.A. Werness, J.M. Huibregtse, A.J. Levine, and P.M. Howley, The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 1990. 63(6): p. 1129-36.
9. Whyte, P., K.J. Buchkovich, J.M. Horowitz, S.H. Friend, M. Raybuck, R.A. Weinberg, and E. Harlow, Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature, 1988. 334(6178): p. 124-9.
10. Linzer, D.I. and A.J. Levine, Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell, 1979. 17(1): p. 43-52.
11. Lane, D.P. and L.V. Crawford, T antigen is bound to a host protein in SV40-transformed cells. Nature, 1979. 278(5701): p. 261-3.
213 214
12. Finlay, C.A., P.W. Hinds, and A.J. Levine, The p53 proto-oncogene can act as a suppressor of transformation. Cell, 1989. 57(7): p. 1083-93.
13. Sarnow, P., P. Hearing, C.W. Anderson, N. Reich, and A.J. Levine, Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix. J Mol Biol, 1982. 162(3): p. 565-83.
14. Dyson, N., P.M. Howley, K. Munger, and E. Harlow, The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science, 1989. 243(4893): p. 934-7.
15. DeCaprio, J.A., J.W. Ludlow, J. Figge, J.Y. Shew, C.M. Huang, W.H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell, 1988. 54(2): p. 275-83.
16. Van den Elsen, P., A. Houweling, and A. Van der Eb, Expression of region E1b of human adenoviruses in the absence of region E1a is not sufficient for complete transformation. Virology, 1983. 128(2): p. 377-90.
17. Lane, D.P., Cancer. p53, guardian of the genome. Nature, 1992. 358(6381): p. 15-6.
18. Horn, H.F. and K.H. Vousden, Coping with stress: multiple ways to activate p53. Oncogene, 2007. 26(9): p. 1306-16.
19. Aylon, Y. and M. Oren, Living with p53, dying of p53. Cell, 2007. 130(4): p. 597-600.
20. Vassilev, L.T., B.T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong, U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi, and E.A. Liu, In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 2004. 303(5659): p. 844-8.
21. Honda, R., H. Tanaka, and H. Yasuda, Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS letters, 1997. 420(1): p. 25-7.
22. Vassilev, L.T., Small-molecule antagonists of p53-MDM2 binding: research tools and potential therapeutics. Cell Cycle, 2004. 3(4): p. 419-21.
23. Klein, C. and L.T. Vassilev, Targeting the p53-MDM2 interaction to treat cancer. Br J Cancer, 2004. 91(8): p. 1415-9.
215
24. Zeimet, A.G., K. Riha, J. Berger, M. Widschwendter, M. Hermann, G. Daxenbichler, and C. Marth, New insights into p53 regulation and gene therapy for cancer. Biochem Pharmacol, 2000. 60(8): p. 1153-63.
25. Buller, R.E., I.B. Runnebaum, B.Y. Karlan, J.A. Horowitz, M. Shahin, T. Buekers, S. Petrauskas, R. Kreienberg, D. Slamon, and M. Pegram, A phase I/II trial of rAd/p53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer gene therapy, 2002. 9(7): p. 553-66.
26. Zeimet, A.G. and C. Marth, Why did p53 gene therapy fail in ovarian cancer? The lancet oncology, 2003. 4(7): p. 415-22.
27. Berk, A.J., Adenovirus promoters and E1A transactivation. Annual review of genetics, 1986. 20: p. 45-79.
28. Querido, E., J.G. Teodoro, and P.E. Branton, Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis. J Virol, 1997. 71(5): p. 3526-33.
29. Steegenga, W.T., N. Riteco, A.G. Jochemsen, F.J. Fallaux, and J.L. Bos, The large E1B protein together with the E4orf6 protein target p53 for active degradation in adenovirus infected cells. Oncogene, 1998. 16(3): p. 349-57.
30. Dobbelstein, M., J. Roth, W.T. Kimberly, A.J. Levine, and T. Shenk, Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence. Embo J, 1997. 16(14): p. 4276-84.
31. Querido, E., R.C. Marcellus, A. Lai, R. Charbonneau, J.G. Teodoro, G. Ketner, and P.E. Branton, Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells. J Virol, 1997. 71(5): p. 3788-98.
32. Querido, E., P. Blanchette, Q. Yan, T. Kamura, M. Morrison, D. Boivin, W.G. Kaelin, R.C. Conaway, J.W. Conaway, and P.E. Branton, Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex. Genes Dev, 2001. 15(23): p. 3104-17.
33. Ying Cheng, C., P. Blanchette, and P.E. Branton, The adenovirus E4orf6 E3 ubiquitin ligase complex assembles in a novel fashion. Virology, 2007. 364(1): p. 36-44.
34. Tollefson, A.E., A. Scaria, T.W. Hermiston, J.S. Ryerse, L.J. Wold, and W.S. Wold, The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells. J Virol, 1996. 70(4): p. 2296-306.
216
35. O'Shea, C.C., C. Soria, B. Bagus, and F. McCormick, Heat shock phenocopies E1B-55K late functions and selectively sensitizes refractory tumor cells to ONYX-015 oncolytic viral therapy. Cancer Cell, 2005. 8(1): p. 61-74.
36. O'Shea, C.C., L. Johnson, B. Bagus, S. Choi, C. Nicholas, A. Shen, L. Boyle, K. Pandey, C. Soria, J. Kunich, Y. Shen, G. Habets, D. Ginzinger, and F. McCormick, Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell, 2004. 6(6): p. 611-23.
37. Soria, C., F.E. Estermann, K.C. Espantman, and C.C. O'Shea, Heterochromatin silencing of p53 target genes by a small viral protein. Nature, 2010. 466(7310): p. 1076-81.
38. Ou, H.D., W. Kwiatkowski, T.J. Deerinck, A. Noske, K.Y. Blain, H.S. Land, C. Soria, C.J. Powers, A.P. May, X. Shu, R.Y. Tsien, J.A. Fitzpatrick, J.A. Long, M.H. Ellisman, S. Choe, and C.C. O'Shea, A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors. Cell, 2012. 151(2): p. 304-19.
39. Stracker, T.H., C.T. Carson, and M.D. Weitzman, Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex. Nature, 2002. 418(6895): p. 348-52.
40. Carvalho, T., J.S. Seeler, K. Ohman, P. Jordan, U. Pettersson, G. Akusjarvi, M. Carmo- Fonseca, and A. Dejean, Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies. J Cell Biol, 1995. 131(1): p. 45-56.
41. Yondola, M.A. and P. Hearing, The adenovirus E4 ORF3 protein binds and reorganizes the TRIM family member transcriptional intermediary factor 1 alpha. J Virol, 2007. 81(8): p. 4264-71.
42. Vink, E.I., M.A. Yondola, K. Wu, and P. Hearing, Adenovirus E4-ORF3-dependent relocalization of TIF1alpha and TIF1gamma relies on access to the Coiled-Coil motif. Virology, 2012. 422(2): p. 317-25.
43. Chirmule, N., K. Propert, S. Magosin, Y. Qian, R. Qian, and J. Wilson, Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther, 1999. 6(9): p. 1574-83.
44. Seshidhar Reddy, P., S. Ganesh, M.P. Limbach, T. Brann, A. Pinkstaff, M. Kaloss, M. Kaleko, and S. Connelly, Development of adenovirus serotype 35 as a gene transfer vector. Virology, 2003. 311(2): p. 384-93.
45. Kakizuka, A., W.H. Miller, Jr., K. Umesono, R.P. Warrell, Jr., S.R. Frankel, V.V. Murty, E. Dmitrovsky, and R.M. Evans, Chromosomal translocation t(15;17) in human acute
217
promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell, 1991. 66(4): p. 663-74.
46. Grignani, F., S. De Matteis, C. Nervi, L. Tomassoni, V. Gelmetti, M. Cioce, M. Fanelli, M. Ruthardt, F.F. Ferrara, I. Zamir, C. Seiser, M.A. Lazar, S. Minucci, and P.G. Pelicci, Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature, 1998. 391(6669): p. 815-8.
47. Melnick, A. and J.D. Licht, Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood, 1999. 93(10): p. 3167-215.
48. Koeffler, H.P. and T.T. Amatruda, 3rd, The effect of retinoids on haemopoiesis--clinical and laboratory studies. Ciba Foundation symposium, 1985. 113: p. 252-67.
49. Breitman, T.R., S.J. Collins, and B.R. Keene, Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid. Blood, 1981. 57(6): p. 1000-4.
50. Chomienne, C., P. Ballerini, M. Huang, M. Cornic, J.P. Abita, S. Castaigne, and L. Degos, In vitro effects of retinoic acid. Nouvelle revue francaise d'hematologie, 1990. 32(1): p. 32-4.
51. Huang, M.E., Y.C. Ye, S.R. Chen, J.R. Chai, J.X. Lu, L. Zhoa, L.J. Gu, and Z.Y. Wang, Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood, 1988. 72(2): p. 567-72.
52. Castaigne, S., C. Chomienne, M.T. Daniel, R. Berger, J.M. Miclea, P. Ballerini, and L. Degos, Retinoic acids in the treatment of acute promyelocytic leukemia. Nouvelle revue francaise d'hematologie, 1990. 32(1): p. 36-8.
53. Wang, Z.G., D. Ruggero, S. Ronchetti, S. Zhong, M. Gaboli, R. Rivi, and P.P. Pandolfi, PML is essential for multiple apoptotic pathways. Nat Genet, 1998. 20(3): p. 266-72.
54. Boutell, C., A. Orr, and R.D. Everett, PML residue lysine 160 is required for the degradation of PML induced by herpes simplex virus type 1 regulatory protein ICP0. J Virol, 2003. 77(16): p. 8686-94.
55. Muller, S. and A. Dejean, Viral immediate-early proteins abrogate the modification by SUMO-1 of PML and Sp100 proteins, correlating with nuclear body disruption. J Virol, 1999. 73(6): p. 5137-43.
218
56. Borden, K.L. and B. Culjkovic, Perspectives in PML: a unifying framework for PML function. Front Biosci, 2009. 14: p. 497-509.
57. Borden, K.L., Pondering the puzzle of PML (promyelocytic leukemia) nuclear bodies: can we fit the pieces together using an RNA regulon? Biochimica et biophysica acta, 2008. 1783(11): p. 2145-54.
58. Ou, H.D., A.P. May, and C.C. O'Shea, The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication. Wiley interdisciplinary reviews. Systems biology and medicine, 2011. 3(1): p. 48-73.
59. Kawai, T. and S. Akira, Regulation of innate immune signalling pathways by the tripartite motif (TRIM) family proteins. EMBO molecular medicine, 2011. 3(9): p. 513-27.
60. Ozato, K., D.M. Shin, T.H. Chang, and H.C. Morse, 3rd, TRIM family proteins and their emerging roles in innate immunity. Nat Rev Immunol, 2008. 8(11): p. 849-60.
61. Uchil, P.D., B.D. Quinlan, W.T. Chan, J.M. Luna, and W. Mothes, TRIM E3 ligases interfere with early and late stages of the retroviral life cycle. PLoS Pathog, 2008. 4(2): p. e16.
62. Carthagena, L., A. Bergamaschi, J.M. Luna, A. David, P.D. Uchil, F. Margottin-Goguet, W. Mothes, U. Hazan, C. Transy, G. Pancino, and S. Nisole, Human TRIM gene expression in response to interferons. PloS one, 2009. 4(3): p. e4894.
63. Napolitano, L.M. and G. Meroni, TRIM family: Pleiotropy and diversification through homomultimer and heteromultimer formation. IUBMB life, 2012. 64(1): p. 64-71.
64. Boudinot, P., L.M. van der Aa, L. Jouneau, L. Du Pasquier, P. Pontarotti, V. Briolat, A. Benmansour, and J.P. Levraud, Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish. PloS one, 2011. 6(7): p. e22022.
65. Meroni, G. and G. Diez-Roux, TRIM/RBCC, a novel class of 'single protein RING finger' E3 ubiquitin ligases. BioEssays : news and reviews in molecular, cellular and developmental biology, 2005. 27(11): p. 1147-57.
66. Deshaies, R.J. and C.A. Joazeiro, RING domain E3 ubiquitin ligases. Annu Rev Biochem, 2009. 78: p. 399-434.
67. Wade, M., Y.V. Wang, and G.M. Wahl, The p53 orchestra: Mdm2 and Mdmx set the tone. Trends Cell Biol, 2010. 20(5): p. 299-309.
219
68. Albor, A., S. El-Hizawi, E.J. Horn, M. Laederich, P. Frosk, K. Wrogemann, and M. Kulesz- Martin, The interaction of Piasy with Trim32, an E3-ubiquitin ligase mutated in limb-girdle muscular dystrophy type 2H, promotes Piasy degradation and regulates UVB-induced keratinocyte apoptosis through NFkappaB. J Biol Chem, 2006. 281(35): p. 25850-66.
69. Gack, M.U., Y.C. Shin, C.H. Joo, T. Urano, C. Liang, L. Sun, O. Takeuchi, S. Akira, Z. Chen, S. Inoue, and J.U. Jung, TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature, 2007. 446(7138): p. 916-920.
70. Agricola, E., R.A. Randall, T. Gaarenstroom, S. Dupont, and C.S. Hill, Recruitment of TIF1gamma to chromatin via its PHD finger-bromodomain activates its ubiquitin ligase and transcriptional repressor activities. Mol Cell, 2011. 43(1): p. 85-96.
71. Tao, H., B.N. Simmons, S. Singireddy, M. Jakkidi, K.M. Short, T.C. Cox, and M.A. Massiah, Structure of the MID1 tandem B-boxes reveals an interaction reminiscent of intermolecular ring heterodimers. Biochemistry, 2008. 47(8): p. 2450-7.
72. Pauling, L. and R.B. Corey, Compound helical configurations of polypeptide chains: structure of proteins of the alpha-keratin type. Nature, 1953. 171(4341): p. 59-61.
73. Lupas, A., Coiled coils: new structures and new functions. Trends Biochem Sci, 1996. 21(10): p. 375-82.
74. Reymond, A., G. Meroni, A. Fantozzi, G. Merla, S. Cairo, L. Luzi, D. Riganelli, E. Zanaria, S. Messali, S. Cainarca, A. Guffanti, S. Minucci, P.G. Pelicci, and A. Ballabio, The tripartite motif family identifies cell compartments. Embo J, 2001. 20(9): p. 2140-51.
75. Short, K.M. and T.C. Cox, Subclassification of the RBCC/TRIM superfamily reveals a novel motif necessary for microtubule binding. J Biol Chem, 2006. 281(13): p. 8970-80.
76. Grutter, C., C. Briand, G. Capitani, P.R. Mittl, S. Papin, J. Tschopp, and M.G. Grutter, Structure of the PRYSPRY-domain: implications for autoinflammatory diseases. FEBS letters, 2006. 580(1): p. 99-106.
77. McNab, F.W., R. Rajsbaum, J.P. Stoye, and A. O'Garra, Tripartite-motif proteins and innate immune regulation. Current opinion in immunology, 2011. 23(1): p. 46-56.
78. Stremlau, M., C.M. Owens, M.J. Perron, M. Kiessling, P. Autissier, and J. Sodroski, The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature, 2004. 427(6977): p. 848-53.
220
79. Yap, M.W., S. Nisole, and J.P. Stoye, A single amino acid change in the SPRY domain of human Trim5alpha leads to HIV-1 restriction. Current biology : CB, 2005. 15(1): p. 73-8.
80. Yang, Y., A. Brandariz-Nunez, T. Fricke, D.N. Ivanov, Z. Sarnak, and F. Diaz-Griffero, Binding of the rhesus TRIM5alpha PRYSPRY domain to capsid is necessary but not sufficient for HIV-1 restriction. Virology, 2014. 448: p. 217-28.
81. Tsai, W.W., Z. Wang, T.T. Yiu, K.C. Akdemir, W. Xia, S. Winter, C.Y. Tsai, X. Shi, D. Schwarzer, W. Plunkett, B. Aronow, O. Gozani, W. Fischle, M.C. Hung, D.J. Patel, and M.C. Barton, TRIM24 links a non-canonical histone signature to breast cancer. Nature, 2010. 468(7326): p. 927-32.
82. Ivanov, A.V., H. Peng, V. Yurchenko, K.L. Yap, D.G. Negorev, D.C. Schultz, E. Psulkowski, W.J. Fredericks, D.E. White, G.G. Maul, M.J. Sadofsky, M.M. Zhou, and F.J. Rauscher, 3rd, PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing. Mol Cell, 2007. 28(5): p. 823-37.
83. Herquel, B., K. Ouararhni, K. Khetchoumian, M. Ignat, M. Teletin, M. Mark, G. Bechade, A. Van Dorsselaer, S. Sanglier-Cianferani, A. Hamiche, F. Cammas, I. Davidson, and R. Losson, Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma. Proc Natl Acad Sci U S A, 2011. 108(20): p. 8212-7.
84. Slack, F.J. and G. Ruvkun, A novel repeat domain that is often associated with RING finger and B-box motifs. Trends Biochem Sci, 1998. 23(12): p. 474-5.
85. Chaudhuri, I., J. Soding, and A.N. Lupas, Evolution of the beta-propeller fold. Proteins, 2008. 71(2): p. 795-803.
86. Fridell, R.A., L.S. Harding, H.P. Bogerd, and B.R. Cullen, Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins. Virology, 1995. 209(2): p. 347-57.
87. Frosk, P., T. Weiler, E. Nylen, T. Sudha, C.R. Greenberg, K. Morgan, T.M. Fujiwara, and K. Wrogemann, Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet, 2002. 70(3): p. 663-72.
88. Schwamborn, J.C., E. Berezikov, and J.A. Knoblich, The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors. Cell, 2009. 136(5): p. 913-25.
221
89. Hammell, C.M., I. Lubin, P.R. Boag, T.K. Blackwell, and V. Ambros, nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell, 2009. 136(5): p. 926-38.
90. Neumuller, R.A., J. Betschinger, A. Fischer, N. Bushati, I. Poernbacher, K. Mechtler, S.M. Cohen, and J.A. Knoblich, Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature, 2008. 454(7201): p. 241-5.
91. Loedige, I., D. Gaidatzis, R. Sack, G. Meister, and W. Filipowicz, The mammalian TRIM- NHL protein TRIM71/LIN-41 is a repressor of mRNA function. Nucleic Acids Res, 2013. 41(1): p. 518-32.
92. Horn, E.J., A. Albor, Y. Liu, S. El-Hizawi, G.E. Vanderbeek, M. Babcock, G.T. Bowden, H. Hennings, G. Lozano, W.C. Weinberg, and M. Kulesz-Martin, RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties. Carcinogenesis, 2004. 25(2): p. 157-67.
93. Pudas, R., T.R. Kiema, P.J. Butler, M. Stewart, and J. Ylanne, Structural basis for vertebrate filamin dimerization. Structure, 2005. 13(1): p. 111-9.
94. Suzuki, M., Y. Hara, C. Takagi, T.S. Yamamoto, and N. Ueno, MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization. Development, 2010. 137(14): p. 2329-39.
95. Lu, T., R. Chen, T.C. Cox, R.X. Moldrich, N. Kurniawan, G. Tan, J.K. Perry, A. Ashworth, P.F. Bartlett, L. Xu, J. Zhang, B. Lu, M. Wu, Q. Shen, Y. Liu, L.J. Richards, and Z. Xiong, X-linked microtubule-associated protein, Mid1, regulates axon development. Proc Natl Acad Sci U S A, 2013. 110(47): p. 19131-6.
96. Skorstengaard, K., M.S. Jensen, P. Sahl, T.E. Petersen, and S. Magnusson, Complete primary structure of bovine plasma fibronectin. European journal of biochemistry / FEBS, 1986. 161(2): p. 441-53.
97. Bouyain, S. and D.J. Leahy, Structure-based mutagenesis of the substrate-recognition domain of Nrdp1/FLRF identifies the binding site for the receptor tyrosine kinase ErbB3. Protein science : a publication of the Protein Society, 2007. 16(4): p. 654-61.
98. Wauman, J., L. De Ceuninck, N. Vanderroost, S. Lievens, and J. Tavernier, RNF41 (Nrdp1) controls type 1 cytokine receptor degradation and ectodomain shedding. J Cell Sci, 2011. 124(Pt 6): p. 921-32.
99. Woodsmith, J., R.C. Jenn, and C.M. Sanderson, Systematic analysis of dimeric E3-RING interactions reveals increased combinatorial complexity in human ubiquitination networks. Molecular & cellular proteomics : MCP, 2012. 11(7): p. M111 016162.
222
100. Sardiello, M., S. Cairo, B. Fontanella, A. Ballabio, and G. Meroni, Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties. BMC evolutionary biology, 2008. 8: p. 225.
101. Rajsbaum, R., J.P. Stoye, and A. O'Garra, Type I interferon-dependent and -independent expression of tripartite motif proteins in immune cells. European journal of immunology, 2008. 38(3): p. 619-30.
102. Barr, S.D., J.R. Smiley, and F.D. Bushman, The interferon response inhibits HIV particle production by induction of TRIM22. PLoS Pathog, 2008. 4(2): p. e1000007.
103. Belloni, E., M. Trubia, P. Gasparini, C. Micucci, C. Tapinassi, S. Confalonieri, P. Nuciforo, B. Martino, F. Lo-Coco, P.G. Pelicci, and P.P. Di Fiore, 8p11 myeloproliferative syndrome with a novel t(7;8) translocation leading to fusion of the FGFR1 and TIF1 genes. Genes Chromosomes Cancer, 2005. 42(3): p. 320-5.
104. Klugbauer, S. and H.M. Rabes, The transcription coactivator HTIF1 and a related protein are fused to the RET receptor tyrosine kinase in childhood papillary thyroid carcinomas. Oncogene, 1999. 18(30): p. 4388-93.
105. Le Douarin, B., C. Zechel, J.M. Garnier, Y. Lutz, L. Tora, P. Pierrat, D. Heery, H. Gronemeyer, P. Chambon, and R. Losson, The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. Embo J, 1995. 14(9): p. 2020-33.
106. Gandini, D., C. De Angeli, G. Aguiari, E. Manzati, F. Lanza, P.P. Pandolfi, A. Cuneo, G.L. Castoldi, and L. del Senno, Preferential expression of the transcription coactivator HTIF1alpha gene in acute myeloid leukemia and MDS-related AML. Leukemia, 2002. 16(5): p. 886-93.
107. Khetchoumian, K., M. Teletin, J. Tisserand, M. Mark, B. Herquel, M. Ignat, J. Zucman- Rossi, F. Cammas, T. Lerouge, C. Thibault, D. Metzger, P. Chambon, and R. Losson, Loss of Trim24 (Tif1alpha) gene function confers oncogenic activity to retinoic acid receptor alpha. Nat Genet, 2007. 39(12): p. 1500-6.
108. Aucagne, R., N. Droin, J. Paggetti, B. Lagrange, A. Largeot, A. Hammann, A. Bataille, L. Martin, K.P. Yan, P. Fenaux, R. Losson, E. Solary, J.N. Bastie, and L. Delva, Transcription intermediary factor 1gamma is a tumor suppressor in mouse and human chronic myelomonocytic leukemia. The Journal of clinical investigation, 2011. 121(6): p. 2361-70.
109. Vincent, D.F., J. Gout, N. Chuvin, V. Arfi, R.M. Pommier, P. Bertolino, N. Jonckheere, D. Ripoche, B. Kaniewski, S. Martel, J.B. Langlois, S. Goddard-Leon, A. Colombe, M. Janier, I. Van Seuningen, R. Losson, U. Valcourt, I. Treilleux, P. Dubus, N. Bardeesy,
223
and L. Bartholin, Tif1gamma suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway. Am J Pathol, 2012. 180(6): p. 2214-21.
110. Takahashi, M., J. Ritz, and G.M. Cooper, Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell, 1985. 42(2): p. 581-8.
111. Kato, M., T. Iwashita, A.A. Akhand, W. Liu, K. Takeda, K. Takeuchi, M. Yoshihara, K. Hossain, J. Wu, J. Du, C. Oh, Y. Kawamoto, H. Suzuki, M. Takahashi, and I. Nakashima, Molecular mechanism of activation and superactivation of Ret tyrosine kinases by ultraviolet light irradiation. Antioxidants & redox signaling, 2000. 2(4): p. 841-9.
112. Dho, S.H. and K.S. Kwon, The Ret finger protein induces apoptosis via its RING finger-B box-coiled-coil motif. J Biol Chem, 2003. 278(34): p. 31902-8.
113. Sato, T., F. Okumura, S. Kano, T. Kondo, T. Ariga, and S. Hatakeyama, TRIM32 promotes neural differentiation through retinoic acid receptor-mediated transcription. J Cell Sci, 2011. 124(Pt 20): p. 3492-502.
114. Frank, D.J., B.A. Edgar, and M.B. Roth, The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis. Development, 2002. 129(2): p. 399-407.
115. Allton, K., A.K. Jain, H.M. Herz, W.W. Tsai, S.Y. Jung, J. Qin, A. Bergmann, R.L. Johnson, and M.C. Barton, Trim24 targets endogenous p53 for degradation. Proc Natl Acad Sci U S A, 2009. 106(28): p. 11612-6.
116. Wang, C., A. Ivanov, L. Chen, W.J. Fredericks, E. Seto, F.J. Rauscher, 3rd, and J. Chen, MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation. Embo J, 2005. 24(18): p. 3279-90.
117. Sho, T., T. Tsukiyama, T. Sato, T. Kondo, J. Cheng, T. Saku, M. Asaka, and S. Hatakeyama, TRIM29 negatively regulates p53 via inhibition of Tip60. Biochimica et biophysica acta, 2011. 1813(6): p. 1245-53.
118. Yuan, Z., A. Villagra, L. Peng, D. Coppola, M. Glozak, E.M. Sotomayor, J. Chen, W.S. Lane, and E. Seto, The ATDC (TRIM29) protein binds p53 and antagonizes p53- mediated functions. Mol Cell Biol, 2010. 30(12): p. 3004-15.
119. Liu, J., B. Welm, K.M. Boucher, M.T. Ebbert, and P.S. Bernard, TRIM29 functions as a tumor suppressor in nontumorigenic breast cells and invasive ER+ breast cancer. Am J Pathol, 2012. 180(2): p. 839-47.
224
120. Wang, L., D.G. Heidt, C.J. Lee, H. Yang, C.D. Logsdon, L. Zhang, E.R. Fearon, M. Ljungman, and D.M. Simeone, Oncogenic function of ATDC in pancreatic cancer through Wnt pathway activation and beta-catenin stabilization. Cancer Cell, 2009. 15(3): p. 207- 19.
121. Obad, S., H. Brunnstrom, J. Vallon-Christersson, A. Borg, K. Drott, and U. Gullberg, Staf50 is a novel p53 target gene conferring reduced clonogenic growth of leukemic U- 937 cells. Oncogene, 2004. 23(23): p. 4050-9.
122. Levine, A.J., The common mechanisms of transformation by the small DNA tumor viruses: The inactivation of tumor suppressor gene products: p53. Virology, 2008.
123. Vogelstein, B., D. Lane, and A.J. Levine, Surfing the p53 network. Nature, 2000. 408(6810): p. 307-10.
124. Vousden, K.H. and C. Prives, Blinded by the Light: The Growing Complexity of p53. Cell, 2009. 137(3): p. 413-31.
125. Kubbutat, M.H., S.N. Jones, and K.H. Vousden, Regulation of p53 stability by Mdm2. Nature, 1997. 387(6630): p. 299-303.
126. Lowe, S.W. and H.E. Ruley, Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev, 1993. 7(4): p. 535-45.
127. Lakin, N.D. and S.P. Jackson, Regulation of p53 in response to DNA damage. Oncogene, 1999. 18(53): p. 7644-55.
128. Sherr, C.J., Divorcing ARF and p53: an unsettled case. Nat Rev Cancer, 2006. 6(9): p. 663-73.
129. Chen, X., L.J. Ko, L. Jayaraman, and C. Prives, p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev, 1996. 10(19): p. 2438-51.
130. Lane, D.P., Exploiting the p53 pathway for the diagnosis and therapy of human cancer. Cold Spring Harb Symp Quant Biol, 2005. 70: p. 489-97.
131. Bischoff, J.R., D.H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J.A. Nye, A. Sampson-Johannes, A. Fattaey, and F. McCormick, An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science, 1996. 274(5286): p. 373-6.
225
132. Debbas, M. and E. White, Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev, 1993. 7(4): p. 546-54.
133. Harada, J.N., A. Shevchenko, D.C. Pallas, and A.J. Berk, Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery. J Virol, 2002. 76(18): p. 9194-206.
134. Berk, A.J., Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus. Oncogene, 2005. 24(52): p. 7673-85.
135. Barker, D.D. and A.J. Berk, Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology, 1987. 156(1): p. 107-21.
136. Khuri, F.R., J. Nemunaitis, I. Ganly, J. Arseneau, I.F. Tannock, L. Romel, M. Gore, J. Ironside, R.H. MacDougall, C. Heise, B. Randlev, A.M. Gillenwater, P. Bruso, S.B. Kaye, W.K. Hong, and D.H. Kirn, a controlled trial of intratumoral ONYX-015, a selectively- replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer [see comments]. Nat Med, 2000. 6(8): p. 879-85.
137. McCormick, F., Cancer-specific viruses and the development of ONYX-015. Cancer Biol Ther, 2003. 2(4 Suppl 1): p. S157-60.
138. O'Shea C, C., L. Johnson, B. Bagus, S. Choi, C. Nicholas, A. Shen, L. Boyle, K. Pandey, C. Soria, J. Kunich, Y. Shen, G. Habets, D. Ginzinger, and F. McCormick, Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity. Cancer Cell, 2004. 6(6): p. 611-23.
139. O'Shea C, C., C. Soria, B. Bagus, and F. McCormick, Heat shock phenocopies E1B-55K late functions and selectively sensitizes refractory tumor cells to ONYX-015 oncolytic viral therapy. Cancer Cell, 2005. 8(1): p. 61-74.
140. Ries, S.J., C.H. Brandts, A.S. Chung, C.H. Biederer, B.C. Hann, E.M. Lipner, F. McCormick, and W.M. Korn, Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015). Nat Med, 2000. 6(10): p. 1128-33.
141. Stott, F.J., S. Bates, M.C. James, B.B. McConnell, M. Starborg, S. Brookes, I. Palmero, K. Ryan, E. Hara, K.H. Vousden, and G. Peters, The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. Embo J, 1998. 17(17): p. 5001-14.
226
142. Olsson, A., C. Manzl, A. Strasser, and A. Villunger, How important are post-translational modifications in p53 for selectivity in target-gene transcription and tumour suppression? Cell Death Differ, 2007. 14(9): p. 1561-75.
143. Khuri, F.R., J. Nemunaitis, I. Ganly, J. Arseneau, I.F. Tannock, L. Romel, M. Gore, J. Ironside, R.H. MacDougall, C. Heise, B. Randlev, A.M. Gillenwater, P. Bruso, S.B. Kaye, W.K. Hong, and D.H. Kirn, a controlled trial of intratumoral ONYX-015, a selectively- replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med, 2000. 6(8): p. 879-85.
144. Bourdon, J.C., K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D.P. Xirodimas, M.K. Saville, and D.P. Lane, p53 isoforms can regulate p53 transcriptional activity. Genes Dev, 2005. 19(18): p. 2122-37.
145. Espinosa, J.M., R.E. Verdun, and B.M. Emerson, p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage. Mol Cell, 2003. 12(4): p. 1015-27.
146. Gannon, J.V., R. Greaves, R. Iggo, and D.P. Lane, Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. Embo J, 1990. 9(5): p. 1595-602.
147. el-Deiry, W.S., S.E. Kern, J.A. Pietenpol, K.W. Kinzler, and B. Vogelstein, Definition of a consensus binding site for p53. Nat Genet, 1992. 1(1): p. 45-9.
148. Kouzarides, T., Chromatin modifications and their function. Cell, 2007. 128(4): p. 693- 705.
149. Bachman, K.E., B.H. Park, I. Rhee, H. Rajagopalan, J.G. Herman, S.B. Baylin, K.W. Kinzler, and B. Vogelstein, Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene. Cancer Cell, 2003. 3(1): p. 89-95.
150. Peters, A.H., D. O'Carroll, H. Scherthan, K. Mechtler, S. Sauer, C. Schofer, K. Weipoltshammer, M. Pagani, M. Lachner, A. Kohlmaier, S. Opravil, M. Doyle, M. Sibilia, and T. Jenuwein, Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell, 2001. 107(3): p. 323-37.
151. Rice, J.C., S.D. Briggs, B. Ueberheide, C.M. Barber, J. Shabanowitz, D.F. Hunt, Y. Shinkai, and C.D. Allis, Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains. Mol Cell, 2003. 12(6): p. 1591-8.
152. Wang, H., W. An, R. Cao, L. Xia, H. Erdjument-Bromage, B. Chatton, P. Tempst, R.G. Roeder, and Y. Zhang, mAM facilitates conversion by ESET of dimethyl to trimethyl
227
lysine 9 of histone H3 to cause transcriptional repression. Mol Cell, 2003. 12(2): p. 475- 87.
153. Tachibana, M., K. Sugimoto, M. Nozaki, J. Ueda, T. Ohta, M. Ohki, M. Fukuda, N. Takeda, H. Niida, H. Kato, and Y. Shinkai, G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev, 2002. 16(14): p. 1779-91.
154. du Chene, I., E. Basyuk, Y.L. Lin, R. Triboulet, A. Knezevich, C. Chable-Bessia, C. Mettling, V. Baillat, J. Reynes, P. Corbeau, E. Bertrand, A. Marcello, S. Emiliani, R. Kiernan, and M. Benkirane, Suv39H1 and HP1gamma are responsible for chromatin- mediated HIV-1 transcriptional silencing and post-integration latency. Embo J, 2007. 26(2): p. 424-35.
155. Neumann, B., M. Held, U. Liebel, H. Erfle, P. Rogers, R. Pepperkok, and J. Ellenberg, High-throughput RNAi screening by time-lapse imaging of live human cells. Nat Methods, 2006. 3(5): p. 385-90.
156. Ferrari, R., M. Pellegrini, G.A. Horwitz, W. Xie, A.J. Berk, and S.K. Kurdistani, Epigenetic reprogramming by adenovirus e1a. Science, 2008. 321(5892): p. 1086-8.
157. Horwitz, G.A., K. Zhang, M.A. McBrian, M. Grunstein, S.K. Kurdistani, and A.J. Berk, Adenovirus small e1a alters global patterns of histone modification. Science, 2008. 321(5892): p. 1084-5.
158. Miller, D.L., C.L. Myers, B. Rickards, H.A. Coller, and S.J. Flint, Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival. Genome Biol, 2007. 8(4): p. R58.
159. O'Shea, C.C., Viruses - seeking and destroying the tumor program. Oncogene, 2005. 24(52): p. 7640-55.
160. Doucas, V., A.M. Ishov, A. Romo, H. Juguilon, M.D. Weitzman, R.M. Evans, and G.G. Maul, Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure. Genes Dev, 1996. 10(2): p. 196-207.
161. Martens, J.H., R.J. O'Sullivan, U. Braunschweig, S. Opravil, M. Radolf, P. Steinlein, and T. Jenuwein, The profile of repeat-associated histone lysine methylation states in the mouse epigenome. Embo J, 2005. 24(4): p. 800-12.
162. Johnson, L., A. Shen, L. Boyle, J. Kunich, K. Pandey, M. Lemmon, T. Hermiston, M. Giedlin, F. McCormick, and A. Fattaey, Selectively replicating adenoviruses targeting
228
deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell, 2002. 1(4): p. 325-37.
163. Jones, N. and T. Shenk, An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A, 1979. 76(8): p. 3665-9.
164. Shepard, R.N. and D.A. Ornelles, E4orf3 is necessary for enhanced S-phase replication of cell cycle-restricted subgroup C adenoviruses. J Virol, 2003. 77(15): p. 8593-5.
165. Halbert, D.N., J.R. Cutt, and T. Shenk, Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff, in J Virol1985. p. 250-7.
166. Cutt, J.R., T. Shenk, and P. Hearing, Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells. J Virol, 1987. 61(2): p. 543-52.
167. Huang, M.M. and P. Hearing, Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection. J Virol, 1989. 63(6): p. 2605-15.
168. Marcellus, R.C., J.G. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton, Adenovirus type 5 early region 4 is responsible for E1A-induced p53- independent apoptosis. J Virol, 1996. 70(9): p. 6207-15.
169. Maehara, K., K. Yamakoshi, N. Ohtani, Y. Kubo, A. Takahashi, S. Arase, N. Jones, and E. Hara, Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53. J Cell Biol, 2005. 168(4): p. 553-60.
170. Eberhardy, S.R. and P.J. Farnham, c-Myc mediates activation of the cad promoter via a post-RNA polymerase II recruitment mechanism. J Biol Chem, 2001. 276(51): p. 48562- 71.
171. Downey, T., Analysis of a multifactor microarray study using Partek genomics solution. Methods Enzymol, 2006. 411: p. 256-70.
172. Bolstad, B.M., R.A. Irizarry, M. Astrand, and T.P. Speed, A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics, 2003. 19(2): p. 185-93.
173. Irizarry, R.A., B.M. Bolstad, F. Collin, L.M. Cope, B. Hobbs, and T.P. Speed, Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res, 2003. 31(4): p. e15.
229
174. Berriz, G.F., O.D. King, B. Bryant, C. Sander, and F.P. Roth, Characterizing gene sets with FuncAssociate. Bioinformatics, 2003. 19(18): p. 2502-4.
175. Yan, B., X. Yang, T.L. Lee, J. Friedman, J. Tang, C. Van Waes, and Z. Chen, Genome- wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-kappaB and other signal transcription factors in head and neck squamous cell carcinoma. Genome Biol, 2007. 8(5): p. R78.
176. Quandt, K., K. Frech, H. Karas, E. Wingender, and T. Werner, MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res, 1995. 23(23): p. 4878-84.
177. Hoh, J., S. Jin, T. Parrado, J. Edington, A.J. Levine, and J. Ott, The p53MH algorithm and its application in detecting p53-responsive genes. Proc Natl Acad Sci U S A, 2002. 99(13): p. 8467-72.
178. Stanley, W.M., Isolation of a Crystalline Protein Possessing the Properties of Tobacco- Mosaic Virus. Science, 1935. 81(2113): p. 644-5.
179. Parato, K.A., D. Senger, P.A. Forsyth, and J.C. Bell, Recent progress in the battle between oncolytic viruses and tumours. Nature reviews. Cancer, 2005. 5(12): p. 965-76.
180. Wilson, J.M., Adenoviruses as gene-delivery vehicles. The New England journal of medicine, 1996. 334(18): p. 1185-7.
181. Fukazawa, T., J. Matsuoka, T. Yamatsuji, Y. Maeda, M.L. Durbin, and Y. Naomoto, Adenovirus-mediated cancer gene therapy and virotherapy (Review). International journal of molecular medicine, 2010. 25(1): p. 3-10.
182. Hollstein, M., D. Sidransky, B. Vogelstein, and C.C. Harris, p53 mutations in human cancers. Science, 1991. 253(5015): p. 49-53.
183. Sarnow, P., Y.S. Ho, J. Williams, and A.J. Levine, Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell, 1982. 28(2): p. 387-94.
184. Lechner, M.S., D.H. Mack, A.B. Finicle, T. Crook, K.H. Vousden, and L.A. Laimins, Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription. Embo J, 1992. 11(8): p. 3045-52.
230
185. Lin, J., J. Chen, B. Elenbaas, and A.J. Levine, Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes Dev, 1994. 8(10): p. 1235-46.
186. Heise, C., A. Sampson-Johannes, A. Williams, F. McCormick, D.D. Von Hoff, and D.H. Kirn, ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med, 1997. 3(6): p. 639-45.
187. Wadell, G., M.L. Hammarskjold, G. Winberg, T.M. Varsanyi, and G. Sundell, Genetic variability of adenoviruses. Annals of the New York Academy of Sciences, 1980. 354: p. 16-42.
188. Green, M., J.K. Mackey, W.S. Wold, and P. Rigden, Thirty-one human adenovirus serotypes (Ad1-Ad31) form five groups (A-E) based upon DNA genome homologies. Virology, 1979. 93(2): p. 481-92.
189. Garon, C.F., K.W. Berry, J.C. Hierholzer, and J.A. Rose, Mapping of base sequence heterologies between genomes from different adenovirus serotypes. Virology, 1973. 54(2): p. 414-26.
190. Hierholzer, J.C., Further subgrouping of the human adenoviruses by differential hemagglutination. The Journal of infectious diseases, 1973. 128(4): p. 541-50.
191. Wadell, G., Classification of human adenoviruses by SDS-polyacrylamide gel electrophoresis of structural polypeptides. Intervirology, 1979. 11(1): p. 47-57.
192. Vogels, R., D. Zuijdgeest, R. van Rijnsoever, E. Hartkoorn, I. Damen, M.P. de Bethune, S. Kostense, G. Penders, N. Helmus, W. Koudstaal, M. Cecchini, A. Wetterwald, M. Sprangers, A. Lemckert, O. Ophorst, B. Koel, M. van Meerendonk, P. Quax, L. Panitti, J. Grimbergen, A. Bout, J. Goudsmit, and M. Havenga, Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol, 2003. 77(15): p. 8263- 71.
193. Wadell, G., Molecular epidemiology of human adenoviruses. Curr Top Microbiol Immunol, 1984. 110: p. 191-220.
194. Haj-Ahmad, Y. and F.L. Graham, Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. J Virol, 1986. 57(1): p. 267-74.
231
195. Stratford-Perricaudet, L.D., I. Makeh, M. Perricaudet, and P. Briand, Widespread long- term gene transfer to mouse skeletal muscles and heart. The Journal of clinical investigation, 1992. 90(2): p. 626-30.
196. Longo, L., E. Donti, A. Mencarelli, G. Avanzi, L. Pegoraro, G. Alimena, A. Tabilio, G. Venti, F. Grignani, and P.G. Pelicci, Mapping of chromosome 17 breakpoints in acute myeloid leukemias. Oncogene, 1990. 5(10): p. 1557-63.
197. Kastner, P., A. Perez, Y. Lutz, C. Rochette-Egly, M.P. Gaub, B. Durand, M. Lanotte, R. Berger, and P. Chambon, Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins. Embo J, 1992. 11(2): p. 629-42.
198. van Wageningen, S., M.C. Breems-de Ridder, J. Nigten, G. Nikoloski, C.A. Erpelinck- Verschueren, B. Lowenberg, T. de Witte, D.G. Tenen, B.A. van der Reijden, and J.H. Jansen, Gene transactivation without direct DNA binding defines a novel gain-of-function for PML-RARalpha. Blood, 2008. 111(3): p. 1634-43.
199. Matsushita, H., P.P. Scaglioni, M. Bhaumik, E.M. Rego, L.F. Cai, S.M. Majid, H. Miyachi, A. Kakizuka, W.H. Miller, Jr., and P.P. Pandolfi, In vivo analysis of the role of aberrant histone deacetylase recruitment and RAR alpha blockade in the pathogenesis of acute promyelocytic leukemia. The Journal of experimental medicine, 2006. 203(4): p. 821-8.
200. Kumar, P.P., O. Bischof, P.K. Purbey, D. Notani, H. Urlaub, A. Dejean, and S. Galande, Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus. Nat Cell Biol, 2007. 9(1): p. 45-56.
201. Doucas, V., The promyelocytic (PML) nuclear compartment and transcription control. Biochem Pharmacol, 2000. 60(8): p. 1197-201.
202. Cohen, N., M. Sharma, A. Kentsis, J.M. Perez, S. Strudwick, and K.L. Borden, PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA. Embo J, 2001. 20(16): p. 4547-59.
203. Bernardi, R. and P.P. Pandolfi, Role of PML and the PML-nuclear body in the control of programmed cell death. Oncogene, 2003. 22(56): p. 9048-57.
204. Pearson, M., R. Carbone, C. Sebastiani, M. Cioce, M. Fagioli, S. Saito, Y. Higashimoto, E. Appella, S. Minucci, P.P. Pandolfi, and P.G. Pelicci, PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature, 2000. 406(6792): p. 207- 10.
232
205. Fogal, V., M. Gostissa, P. Sandy, P. Zacchi, T. Sternsdorf, K. Jensen, P.P. Pandolfi, H. Will, C. Schneider, and G. Del Sal, Regulation of p53 activity in nuclear bodies by a specific PML isoform. Embo J, 2000. 19(22): p. 6185-95.
206. Guo, A., P. Salomoni, J. Luo, A. Shih, S. Zhong, W. Gu, and P.P. Pandolfi, The function of PML in p53-dependent apoptosis. Nat Cell Biol, 2000. 2(10): p. 730-6.
207. Khetchoumian, K., M. Teletin, J. Tisserand, B. Herquel, K. Ouararhni, and R. Losson, Trim24 (Tif1 alpha): an essential 'brake' for retinoic acid-induced transcription to prevent liver cancer. Cell Cycle, 2008. 7(23): p. 3647-52.
208. Tisserand, J., K. Khetchoumian, C. Thibault, D. Dembele, P. Chambon, and R. Losson, Tripartite motif 24 (Trim24/Tif1alpha) tumor suppressor protein is a novel negative regulator of interferon (IFN)/signal transducers and activators of transcription (STAT) signaling pathway acting through retinoic acid receptor alpha (Raralpha) inhibition. J Biol Chem, 2011. 286(38): p. 33369-79.
209. Le Douarin, B., A.L. Nielsen, J.M. Garnier, H. Ichinose, F. Jeanmougin, R. Losson, and P. Chambon, A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. Embo J, 1996. 15(23): p. 6701-15.
210. Kikuchi, M., F. Okumura, T. Tsukiyama, M. Watanabe, N. Miyajima, J. Tanaka, M. Imamura, and S. Hatakeyama, TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells. Biochimica et biophysica acta, 2009. 1793(12): p. 1828-36.
211. He, W., D.C. Dorn, H. Erdjument-Bromage, P. Tempst, M.A. Moore, and J. Massague, Hematopoiesis controlled by distinct TIF1gamma and Smad4 branches of the TGFbeta pathway. Cell, 2006. 125(5): p. 929-41.
212. Morsut, L., K.P. Yan, E. Enzo, M. Aragona, S.M. Soligo, O. Wendling, M. Mark, K. Khetchoumian, G. Bressan, P. Chambon, S. Dupont, R. Losson, and S. Piccolo, Negative control of Smad activity by ectodermin/Tif1gamma patterns the mammalian embryo. Development, 2010. 137(15): p. 2571-8.
213. Vincent, D.F., K.P. Yan, I. Treilleux, F. Gay, V. Arfi, B. Kaniewski, J.C. Marie, F. Lepinasse, S. Martel, S. Goddard-Leon, J.L. Iovanna, P. Dubus, S. Garcia, A. Puisieux, R. Rimokh, N. Bardeesy, J.Y. Scoazec, R. Losson, and L. Bartholin, Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas. PLoS genetics, 2009. 5(7): p. e1000575.
214. Forrester, N.A., R.N. Patel, T. Speiseder, P. Groitl, G.G. Sedgwick, N.J. Shimwell, R.I. Seed, P.O. Catnaigh, C.J. McCabe, G.S. Stewart, T. Dobner, R.J. Grand, A. Martin, and A.S. Turnell, Adenovirus E4orf3 targets transcriptional intermediary factor 1gamma for proteasome-dependent degradation during infection. J Virol, 2012. 86(6): p. 3167-79.
233
215. Stracker, T.H., D.V. Lee, C.T. Carson, F.D. Araujo, D.A. Ornelles, and M.D. Weitzman, Serotype-specific reorganization of the Mre11 complex by adenoviral E4orf3 proteins. J Virol, 2005. 79(11): p. 6664-73.
216. Forrester, N.A., G.G. Sedgwick, A. Thomas, A.N. Blackford, T. Speiseder, T. Dobner, P.J. Byrd, G.S. Stewart, A.S. Turnell, and R.J. Grand, Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection. J Virol, 2011. 85(5): p. 2201-11.
217. Williams, R.S., J.S. Williams, and J.A. Tainer, Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem Cell Biol, 2007. 85(4): p. 509-20.
218. Carson, C.T., N.I. Orazio, D.V. Lee, J. Suh, S. Bekker-Jensen, F.D. Araujo, S.S. Lakdawala, C.E. Lilley, J. Bartek, J. Lukas, and M.D. Weitzman, Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection. Embo J, 2009. 28(6): p. 652-62.
219. Evans, J.D. and P. Hearing, Distinct roles of the Adenovirus E4 ORF3 protein in viral DNA replication and inhibition of genome concatenation. J Virol, 2003. 77(9): p. 5295- 304.
220. Ullman, A.J. and P. Hearing, Cellular proteins PML and Daxx mediate an innate antiviral defense antagonized by the adenovirus E4 ORF3 protein. J Virol, 2008. 82(15): p. 7325- 35.
221. Kao, C.C., P.R. Yew, and A.J. Berk, Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins. Virology, 1990. 179(2): p. 806- 14.
222. Nwanegbo, E., E. Vardas, W. Gao, H. Whittle, H. Sun, D. Rowe, P.D. Robbins, and A. Gambotto, Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clinical and diagnostic laboratory immunology, 2004. 11(2): p. 351-7.
223. Soria, C., F.E. Estermann, K.C. Espantman, and C.C. O'Shea, Heterochromatin silencing of p53 target genes by a small viral protein. Nature. 466(7310): p. 1076-81.
224. Larkin, M.A., G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, and D.G. Higgins, Clustal W and Clustal X version 2.0. Bioinformatics, 2007. 23(21): p. 2947-8.
234
225. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar, MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution, 2011. 28(10): p. 2731-9.
226. Jones, N. and T. Shenk, Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell, 1979. 17(3): p. 683-9.
227. Shepard, R.N. and D.A. Ornelles, Diverse roles for E4orf3 at late times of infection revealed in an E1B 55-kilodalton protein mutant background. J Virol, 2004. 78(18): p. 9924-35.
228. Walhout, A.J., G.F. Temple, M.A. Brasch, J.L. Hartley, M.A. Lorson, S. van den Heuvel, and M. Vidal, GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods in enzymology, 2000. 328: p. 575-92.
229. Sarnow, P., C.A. Sullivan, and A.J. Levine, A monoclonal antibody detecting the adenovirus type 5-E1b-58Kd tumor antigen: characterization of the E1b-58Kd tumor antigen in adenovirus-infected and -transformed cells. Virology, 1982. 120(2): p. 510-7.
230. Schneider, C.A., W.S. Rasband, and K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis. Nature methods, 2012. 9(7): p. 671-5.
231. Rajsbaum, R., A. Garcia-Sastre, and G.A. Versteeg, TRIMmunity: The Roles of the TRIM E3-Ubiquitin Ligase Family in Innate Antiviral Immunity. J Mol Biol, 2013.
232. Sakuma, R., J.A. Noser, S. Ohmine, and Y. Ikeda, Rhesus monkey TRIM5alpha restricts HIV-1 production through rapid degradation of viral Gag polyproteins. Nat Med, 2007. 13(5): p. 631-5.
233. Leppard, K.N. and R.D. Everett, The adenovirus type 5 E1b 55K and E4 Orf3 proteins associate in infected cells and affect ND10 components. J Gen Virol, 1999. 80 ( Pt 4): p. 997-1008.
234. Bellacchio, E. and M.G. Paggi, Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery. J Cell Physiol, 2013. 228(2): p. 285-91.
235. Everett, R.D., C. Parada, P. Gripon, H. Sirma, and A. Orr, Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100. J Virol, 2008. 82(6): p. 2661-72.
235
236. Bailey, B., D.L. Farkas, D.L. Taylor, and F. Lanni, Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature, 1993. 366(6450): p. 44-8.
237. Ahn, J.H. and G.S. Hayward, The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells. J Virol, 1997. 71(6): p. 4599-613.
238. Lee, H.R., D.J. Kim, J.M. Lee, C.Y. Choi, B.Y. Ahn, G.S. Hayward, and J.H. Ahn, Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells. J Virol, 2004. 78(12): p. 6527-42.
239. Ahn, J.H., W.J. Jang, and G.S. Hayward, The human cytomegalovirus IE2 and UL112- 113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10). J Virol, 1999. 73(12): p. 10458-71.
240. Cuchet-Lourenco, D., E. Vanni, M. Glass, A. Orr, and R.D. Everett, Herpes simplex virus 1 ubiquitin ligase ICP0 interacts with PML isoform I and induces its SUMO-independent degradation. J Virol, 2012. 86(20): p. 11209-22.
241. Chu, Y. and X. Yang, SUMO E3 ligase activity of TRIM proteins. Oncogene, 2011. 30(9): p. 1108-16.
242. Mammen M, C.S.-K., Whitesides GM, Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhivitors. Angewandte Chemie International Edition1998.
243. Sommer, M.H., A.L. Scully, and D.H. Spector, Transactivation by the human cytomegalovirus IE2 86-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol, 1994. 68(10): p. 6223-31.