Printed Edition of the Special Issue Published in Viruses Recent CMV Research Edited by Anamaris M. Colberg-Poley www.mdpi.com/journal/viruses Anamaris M. Colberg-Poley (Ed.) Recent CMV Research This book is a reprint of the special issue that appeared in the online open access journal Viruses (ISSN 1999-4915) in 2013 (http://www.mdpi.com/journal/viruses/special_issues/recent_cmv_research). Guest Editor Anamaris M. Colberg-Poley Professor of Integrative Systems Biology and of Pediatrics George Washington University School of Medicine and Health Sciences Senior Investigator, Research Center for Genetic Medicine, Children's National Health System Washington, DC 20010, USA Editorial Office MDPI AG Klybeckstrasse 64 Basel, Switzerland Publisher Shu-Kun Lin Managing Editor Delia Costache 1. Edition 2014 MDPI • Basel • Beijing • Wuhan ISBN 978-3-906980-53-9 (Hbk) ISBN 978-3-906980-54-6 (PDF) © 2014 by the authors; licensee MDPI, Basel, Switzerland. All articles in this volume are Open Access distributed under the Creative Commons License (CC BY), which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. However, the dissemination and distribution of physical copies of this book as a whole is restricted to MDPI, Basel, Switzerland. III Table of Contents Anamaris M. Colberg-Poley Preface Guest Editor.................................................................................................................. VII Kayla Dufrene, Roberta L. DeBiasi and Anamaris M. Colberg-Poley Introduction: Preface of the Special Issue: “Recent CMV Research” Reprinted from: Viruses 2014 , 6 (1), 336-339 www.mdpi.com/1999-4915/6/1/336...............................................................................................1 CMV Pathogenesis and its Control John H. Sinclair and Matthew B. Reeves Human Cytomegalovirus Manipulation of Latently Infected Cells Reprinted from: Viruses 2013 , 5 (11), 2803-2824 www.mdpi.com/1999-4915/5/11/2803 ...........................................................................................5 Martin Zydek, Matthew Petitt, June Fang-Hoover, Barbara Adler, Lawrence M. Kauvar, Lenore Pereira and Takako Tabata HCMV Infection of Human Trophoblast Progenitor Cells of the Placenta Is Neutralized by a Human Monoclonal Antibody to Glycoprotein B and Not by Antibodies to the Pentamer Complex Reprinted from: Viruses 2014 , 6 (3), 1346-1364 www.mdpi.com/1999-4915/6/3/1346........................................................................................... 27 Sonia M. Restrepo-Gualteros, Lina E. Jaramillo-Barberi, Monica Gonzalez Santos, Carlos E. Rodriguez-Martinez, Geovanny F. Perez, Maria J. Gutierrez and Gustavo Nino Characterization of Cytomegalovirus Lung Infection in Non-HIV Infected Children Reprinted from: Viruses 2014 , 6 (5), 2038-2051 www.mdpi.com/1999-4915/6/5/2038........................................................................................... 45 Jesse D. Deere and Peter A. Barry Using the Nonhuman Primate Model of HCMV to Guide Vaccine Development Reprinted from: Viruses 2014 , 6 (4), 1483-1501 www.mdpi.com/1999-4915/6/4/1483........................................................................................... 59 Immune and DNA Damage Responses Linked to CMV Patrick J. Hanley and Catherine M. Bollard Controlling Cytomegalovirus: Helping the Immune System Take the Lead Reprinted from: Viruses 2014 , 6 (6), 2242-2258 www.mdpi.com/1999-4915/6/6/2242........................................................................................... 79 IV Emily V. Stevenson, Donna Collins-McMillen, Jung Heon Kim, Stephen J. Cieply, Gretchen L. Bentz and Andrew D. Yurochko HCMV Reprogramming of Infected Monocyte Survival and Differentiation: A Goldilocks Phenomenon Reprinted from: Viruses 2014 , 6 (2), 782-807 www.mdpi.com/1999-4915/6/2/782............................................................................................. 97 Annette Fink, Angeliqué Renzaho, Matthias J. Reddehase and Niels A. W. Lemmermann The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion Reprinted from: Viruses 2013 , 5 (12), 3171-3191 www.mdpi.com/1999-4915/5/12/3171 ....................................................................................... 123 Annette Fink, Julia K. Büttner, Doris Thomas, Rafaela Holtappels, Matthias J. Reddehase and Niels A. W. Lemmermann Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene Reprinted from: Viruses 2014 , 6 (2), 808-831 www.mdpi.com/1999-4915/6/2/808........................................................................................... 145 Bindu Raghavan, Charles H. Cook and Joanne Trgovcich The Carboxy Terminal Region of the Human Cytomegalovirus Immediate Early 1 (IE1) Protein Disrupts Type II Inteferon Signaling Reprinted from: Viruses 2014 , 6 (4), 1502-1524 www.mdpi.com/1999-4915/6/4/1502......................................................................................... 171 Xiaofei E and Timothy F. Kowalik The DNA Damage Response Induced by Infection with Human Cytomegalovirus and Other Viruses Reprinted from: Viruses 2014 , 6 (5), 2155-2185 www.mdpi.com/1999-4915/6/5/2155......................................................................................... 195 Amit S. Kulkarni and Elizabeth A. Fortunato Modulation of Homology-Directed Repair in T98G Glioblastoma Cells Due to Interactions between Wildtype p53, Rad51 and HCMV IE1-72 Reprinted from: Viruses 2014 , 6 (3), 968-985 www.mdpi.com/1999-4915/6/3/968........................................................................................... 227 V The CMV Life Cycle Laura Graf, Rike Webel, Sabrina Wagner, Stuart T. Hamilton, William D. Rawlinson, Heinrich Sticht and Manfred Marschall The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins Reprinted from: Viruses 2013 , 5 (12), 3213-3230 ....................................................................... 245 www.mdpi.com/1999-4915/5/12/3213 Rico Rana and Bonita J. Biegalke Human Cytomegalovirus UL34 Early and Late Proteins Are Essential for Viral Replication Reprinted from: Viruses 2014 , 6 (2), 476-488 ............................................................................. 265 www.mdpi.com/1999-4915/6/2/476 Ina Niemann, Anna Reichel and Thomas Stamminger Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7- trans -Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis Reprinted from: Viruses 2014 , 6 (2), 661-682 ............................................................................. 279 www.mdpi.com/1999-4915/6/2/661 Rebecca Marie Smith, Srivenkat Kosuri and Julie Anne Kerry Role of Human Cytomegalovirus Tegument Proteins in Virion Assembly Reprinted from: Viruses 2014 , 6 (2), 582-605 ............................................................................. 301 www.mdpi.com/1999-4915/6/2/582 Vanessa M. Noriega, Thomas J. Gardner, Veronika Redmann, Gerold Bongers, Sergio A. Lira and Domenico Tortorella Human Cytomegalovirus US28 Facilitates Cell-to-Cell Viral Dissemination Reprinted from: Viruses 2014 , 6 (3), 1202-1218 ......................................................................... 325 www.mdpi.com/1999-4915/6/3/1202 The Use of Novel Technologies Sabine Reyda, Nicole Büscher, Stefan Tenzer and Bodo Plachter Proteomic Analyses of Human Cytomegalovirus Strain AD169 Derivatives Reveal Highly Conserved Patterns of Viral and Cellular Proteins in Infected Fibroblasts Reprinted from: Viruses 2014 , 6 (1), 172-188 ............................................................................. 343 www.mdpi.com/1999-4915/6/1/172 Mark R. Schleiss, Shane McAllister, Anibal G. Armién, Nelmary Hernandez-Alvarado, Claudia Fernández-Alarcón, Jason C. Zabeli, Thiruvarangan Ramaraj, John A. Crow and Michael A. McVoy Molecular and Biological Characterization of a New Isolate of Guinea Pig Cytomegalovirus Reprinted from: Viruses 2014 , 6 (2), 448-475 ............................................................................. 361 www.mdpi.com/1999-4915/6/2/448 VI Josephine S. Gnanandarajah, Peter A. Gillis, Nelmary Hernandez-Alvarado, LeeAnn Higgins, Todd W. Markowski, Heungsup Sung, Sheila Lumley and Mark R. Schleiss Identification by Mass Spectrometry and Immune Response Analysis of Guinea Pig Cytomegalovirus (GPCMV) Pentameric Complex Proteins GP129, 131 and 133 Reprinted from: Viruses 2014 , 6 (2), 727-751 ............................................................................. 391 www.mdpi.com/1999-4915/6/2/727 Francisco Puerta Martínez and Qiyi Tang Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate- Early Protein 1 by Protein Array Assay Reprinted from: Viruses 2014 , 6 (1), 89-105 ............................................................................... 417 www.mdpi.com/1999-4915/6/1/89 Endrit Elbasani, Ildar Gabaev, Lars Steinbrück, Martin Messerle and Eva Maria Borst Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes Reprinted from: Viruses 2014 , 6 (1), 354-370 ............................................................................. 435 www.mdpi.com/1999-4915/6/1/354 Shivaprasad Bhuvanendran, Kyle Salka, Kristin Rainey, Sen Chandra Sreetama, Elizabeth Williams, Margretha Leeker, Vidhya Prasad, Jonathan Boyd, George H. Patterson, Jyoti K. Jaiswal and Anamaris M. Colberg-Poley Superresolution Imaging of Human Cytomegalovirus vMIA Localization in Sub- Mitochondrial Compartments Reprinted from: Viruses 2014 , 6 (4), 1612-1636 ......................................................................... 453 www.mdpi.com/1999-4915/6/4/1612 Steven Sijmons, Marc Van Ranst and Piet Maes Genomic and Functional Characteristics of Human Cytomegalovirus Revealed by Next- Generation Sequencing Reprinted from: Viruses 2014 , 6 (3), 1049-1072 ......................................................................... 479 www.mdpi.com/1999-4915/6/3/1049 Zhu Yang, Gia-Phong Vu, Hua Qian, Yuan-Chuan Chen, Yu Wang, Michael Reeves, Ke Zen and Fenyong Liu Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication Reprinted from: Viruses 2014 , 6 (6), 2376-2391 ......................................................................... 503 www.mdpi.com/1999-4915/6/6/2376 VII Preface In developed countries, human cytomegalovirus (CMV) is the major infectious cause of congenital birth defects including microcephaly, mental retardation, sensorineural hearing loss, and intrauterine growth restriction. Nonetheless, because of the potential for teratogenicity and toxic effects, no prenatal therapeutic treatment is currently approved by the Food and Drug Administration for congenital CMV infection. Therefore there is great interest in understanding CMV growth and blocking or altogether preventing CMV infection. When I received the email from the Viruses Editor-in-Chief, Eric Freed, about editing a Special Issue of Viruses on Recent CMV Research, I debated with myself and my husband (a Pediatric Infectious Disease physician) about the need for another dedicated CMV issue. There were several recent books, chapters and special issues on CMV including the comprehensive “Cytomegaloviruses From Molecular Pathogenesis to Intervention” edited by Matthias Reddehase with the assistance of Niels Lemmerman (Caister Academic Press, 2013) as well as a recent supplement (Supplement 4) of Clinical Infectious Diseases (2013, volume 57) on Prenatal Therapy of Congenital Cytomegalovirus Infection Nonetheless, having participated in the 4 th Congenital CMV Conference in San Francisco and then being interviewed by Kayla Dufrene, a young woman who had suffered congenital CMV infection convinced me of the vital importance of continued efforts to understand the CMV life cycle and for developing new approaches to preventing and treating CMV diseases. With this in mind, I accepted serving as Guest Editor for the Viruses Special Issue on Recent CMV Research. Following my husband’s advice, I asked Kayla if we could use her autobiography, which she wrote following our meeting for her writing class at Gallaudet University, as the Introduction for our Special Issue. She accepted and it is the first paper in this book (Dufrene et al., 2014). Her autobiography is the backdrop for this issue and should serve as inspiration for many CMV investigators. Tellingly, Kayla has also been invited to speak about her life’s story at the 2014 Cytomegalovirus Public Health and Policy Conference in Utah. She provides an unparalleled perspective for the CMV field. I am very pleased with this Viruses Special Issue. Of particular interest to families and caregivers affected by CMV diseases are several papers: addressing prevention of CMV infection of trophoblast cells (Zydek et al., 2014), CMV latency (Sinclair and Reeves, 2013), as well as of CMV lung infections in non-HIV infected children (Restrepo-Gualteros et al., 2014). Our ability to enhance immune responses for controlling CMV infection (Hanley and Bollard, 2014) and new strategies for CMV vaccine development guided by non-human primate studies (Deere and Barry, 2014) are discussed in two excellent reviews. Several articles address the CMV manipulation of the immune system, both innate and adaptive immune responses (Stevenson et al., 2014, Fink et al, 2013, 2014, Raghavan et al., 2014) and of DNA damage responses (E and Kowalik, 2014; Kulkarni and Fortunato, 2014). The CMV life cycle itself is the subject of several papers, including demonstration that the UL97 protein interacts with cell cyclins (Graf et al., 2014), the UL34 early and late proteins VIII are required for infection (Rana and Biegalke, 2014), the intracellular trafficking of CMV proteins (Niemann et al., 2014), the roles of CMV tegument proteins in virion assembly (Smith et al., 2014), and the effects of US28 protein on cell-to-cell spread of CMV (Noriega et al., 2014). Development of new drugs for CMV depends heavily on the generation of novel understanding of CMV lifecycle and the mechanisms it uses in its progression. With this in mind, many studies in this book has made significant contributions of state-of-the-art technologies to the field including the use of mass spectrometry (Reyda et al., 2014; Gnanandarajah et al., 2014), protein arrays (Puerta Martínez and Tang, 2014), adenofection for efficient transfection of CMV genomes (Elbasani et al., 2014), superresolution microscopy (Bhuvanendran et al., 2014), next generation sequencing (Sijmons et al., 2014) and targeted ribozymes (Yang et al., 2014) to enhance our understanding of CMV infection. Finally, I want to thank all the colleagues who contributed papers to this Special Issue, which has impressed the publishers and the readers alike. In fact the publisher selected two of the papers from this special issue (Zydek et al., 2014; Deere and Barry, 2014) to highlight in the MDPI Magazine. It is my hope that this special issue provides a valuable resource for the families and investigators in the CMV field. Anamaris M. Colberg-Poley Guest Editor 1 Reprinted from Viruses . Cite as: Dufrene, K.; DeBiasi, R.L.; Colberg-Poley, A.M. Preface of the Special Issue: “ Recent CMV Research ” Viruses 2014 , 6 , 336-339. Preface Preface of the Special Issue: “ Recent CMV Research ” Kayla Dufrene 1 , Roberta L. DeBiasi 2,3 and Anamaris M. Colberg-Poley 2,4,5, * 1 Gallaudet University, 800 Florida Avenue NE, Washington, DC 20002, USA; E-Mail: kayla.dufrene@gallaudet.edu 2 Departments of Pediatrics, Children’s National Medical Center, 111 Michigan Avenue, NW Washington, DC 20010, USA; E-Mail: rdebiasi@childrensnational.org 3 Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037, USA 4 Integrative Systems Biology, Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Avenue, NW Washington, DC 20010, USA 5 Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA * Author to whom correspondence should be addressed; E-Mail: acolberg-poley@childrensnational.org; Tel.: +1-202-476-3984; Fax: +1-202-476-6014. Received: 9 December 2013 / Accepted: 14 January 2014 / Published: 22 January 2014 1. Foreword This Viruses Special Issue on Recent Cytomegalovirus (CMV) Research is dedicated to the patients who have suffered CMV infection and to their parents, families and caregivers. We are including as a Preface to this issue the insights of a young college student, Kayla Dufrene, who suffered congenital CMV infection and contacted me and Dr. Roberta DeBiasi, to interview us to learn more about CMV. As I was just returning to the DC area from the 4th Congenital CMV Conference in San Francisco, I was particularly receptive to her request. When we met Kayla, we were both impressed with her personal strength and ability to cope with her disabilities and needed medical treatments. Despite it all, Kayla has an exceptionally positive outlook on life, feeling even lucky. She has not only coped, but has transcended her difficulties. I am proud to say that she was on the Dean’s List (Figure 1) at Gallaudet University. Ultimately, her hope lies in our fields’ efforts to develop a vaccine to prevent CMV disease in other children. Her autobiography (in her own words) is our Preface. For those of us who work on the virus and anyone interested in the consequences of CMV disease, it is a touching and inspiring read (Figure 2). 2 Anamaris Colberg-Poley, Guest Editor, Viruses, Special Issue, Recent CMV Research and Roberta L. DeBiasi, Professor of Pediatrics, GWU, Acting Chief, Division of Pediatric Infectious Diseases, Children’s National Medical Center Figure 1. Kayla finding out that she made the Dean’s List at Gallaudet University. 2. Preface: Cytomegalovirus — Patient Monograph — Kayla Dufrene Prior to writing a research paper for a college assignment, I never felt the need to learn more about Cytomegalovirus (CMV). I didn’t know a lot about CM V, I just grew up hearing my Mom tell doctors that it’s what I was born with. It’s the reason why I have hearing loss, bad eyesight, and muscle problems in my legs, and also the cyst in my brain. It’s also why I have had to endure two eye surgeries and surgery on both my hips. When I was born, I was very sick. Besides having CMV I had an enlarged liver (when the liver swells beyond its normal size) and yellow jaundice (yellowing of the skin). I was very tiny and had to stay in the hospital for a month. The doctors told my birth parents I would either die or not have a good quality of life. The decision was then made to put me up for adoption. Figure 2. Kayla enjoying the cherry blossoms. 3 It turns out I definitely DID want to know more about CMV. What are the symptoms? Is there a cure? Is this a genetic disease? Is there genetic testing? How does it affect my body? How does someone get CMV? Is it an STD? How is it diagnosed? Can I get it later in life? Is there a vaccine for infants? Is there a test for CMV? Is it contagious? Who can get CMV? How many people are diagnosed with cytomegalovirus? How has it affected me? Do I still have it in my system? How does my case compare to others? How will it affect me later in life? Could it affect my sex partner? Could I really have died? Did it affect my birth mom? Is it a genetic disease and something I have to worry about in the future? After reading texts and online references on Gallaudet’s library webpage, I had a foundation for my search and I was ready to start building from there. I developed a desire for more knowledge and answers to a more of my questions. I learned that CMV is a more popular subject than I thought. When I read about the impact it can have on the family, it made me understand a little more why my birth parents put me up for adoption. They couldn’t have known the extent that I would be affected yet here I am, 19 years later, a student at Gallaudet University. I was very excited to continue my search and learn more about the disease that has made me the person who I am today. My reading taught me that development of a CMV vaccine was a national top priority and I wanted to learn more about the efforts that doctors and researchers are going through to make a vaccine a reality. I know firsthand the effects of CMV: Knowing a vaccine could have prevented a lot of what I had to go through and could prevent newborns in the future from being affected by CMV I believe is worthwhile. CMV is very common and the effects it can have on a child and the family can be very hard. I know these from my own experiences of being picked on in school for wearing a hearing aid and being “the girl who walked a little funny”. I was excited to read about clinical trials that are testing potential vaccines for CMV. One of the articles explained a clinical trial to develop a vaccine to prevent CMV among mothers and infants. The possibility of a vaccine is very real. CMV has affected my life from the surgeries to the teasing in school and the thought that a vaccine could have prevented all of that is mind-boggling. My life could have turned out differently. My reading sparked my interest in interviewing someone knowledgeable about CMV research and disease. The deadline for having an interviewee was quickly approaching. I thought long and hard about where I could find someone whom I could interview. I then thought that since CMV is found in babies, maybe I should ask a pediatrician. I found out that the Children’s National Medical Center was just a few metro stops from Gallaudet and I was lucky to identify both a scientist (Dr. Colberg-Poley) and a doctor (Dr. Roberta DeBiasi) who focus their careers in this very area. They were both more than happy to meet with me. On the day of the interview, I was really nervous and wanted to make sure I had everything I needed prepared. I explained to them that I was writing a paper on a topic that had to relate to me so I chose CMV. I told them how I was born with CMV and of how it was the reason I had two eye surgeries when I was a kid, a hip surgery on both hips, and why I wear glasses and a hearing aid. They were thrilled to have me there, eager to share their knowledge about CMV, and just as excited to meet and speak with me as I was excited to meet with them. Dr. DeBiasi shared that in her entire career, she had never met someone as an adult who had been diagnosed with CMV as a baby. She said it was a good experience for her and an honor to 4 meet me. I shared many of my frustrations, such as the fact that no one figured out I was deaf until third grade, and I was able to ask if I born deaf or if it was detected late. I learned that even though the virus is latent in my body (just like anyone else who is infected with CMV at any time in their life); it is not something I have to worry about when I have a sexual relationship or children. I learned that it is not a genetic illness, and not something that I am going to pass on to my children genetically. I also learned that it is hard for doctors to accept that there are diseases they can diagnos e, but for which they can’t do anything about, and they want to help change that. The one big thing that I took away was that I’m going to be okay and I do not have to stress about my future like wondering if CMV was going to affect my future children. I also realized that, when my birth mom put me up for adoption, she couldn’t have known what the future held for me. I’m glad that she’s okay. My adoptive mom did a very brave thing of adopting a baby that she knew was sick and never bat an eye and, over the last 19 years, I have not once heard her complain. Hearing Dr. Colberg-Poley and Dr. DeBiasi describe how there are even worse possible outcomes for people with CMV makes me feel extremely lucky. Something so small created a lot of problems for me growing up but it has made me a stronger person and more understanding of other people who have other problems. If it wasn’t for cytomegalovirus I wouldn’t have hearing problems and I wouldn’t be at Gallaudet University and I never would have had the chance to fin d out all of this information about CMV. Doing the interviews was the most beneficial thing to me because I have never had anyone to answer my questions. I was not sure where my search would lead me and what I would find out. I have always lived with the fear of not knowing how Cytomegalovirus would affect my future. I have always had questions like: What are the symptoms? Is there a cure? Is this a genetic disease? Do I still have it in my system? How does my case compare to others? How will it affect me later in life? Could it affect my sex partner? But I never had any answers. Now I have all the answers to all the questions I have ever asked. The search was hard and stressful but I’m glad I had the opportunity to finally learn about CMV. I have now made contact with people of whom I can ask questions that may arise later. During the interviews I learned things that I could not get through any article or any book and I got real answers. It’s amazing that something as small as a tiny virus could have such a huge impact and effect on my life. After my reading and interviews, I left feeling much more knowledgeable about CMV. Dr. Colberg-Poley and DeBiasi answered all of my questions and made me leave with a new-found confidence. I’m going to be okay in the f uture and so will my kids. CMV affected me as a baby and growing up it caused me a lot of problems, but it’s because of having Cytomegalovirus that I’ve had the experiences I’ve had and why I’m at Gallaudet University. When I started my research, I was not sure where it would lead me or what I would find. I have learned a lot about the virus and a lot about myself and just because the past was tough, it doesn’t mean the future has to be. 5 CMV Pathogenesis and its Control Reprinted from Viruses Cite as: Sinclair, J.H.; Reeves, M.B. Human Cytomegalovirus Manipulation of Latently Infected Cells. Viruses 2013 , 5 , 2803-2824. Review Human Cytomegalovirus Manipulation of Latently Infected Cells John H. Sinclair 1 and Matthew B. Reeves 2, * 1 Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 0QQ, UK; E-Mail: js152@cam.ac.uk 2 Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, UK * Author to whom correspondence should be addressed; E-Mail: matthew.reeves@ucl.ac.uk; Tel.: +44-(0)207-794-0500 (ext. 33109). Received: 17 October 2013; in revised form: 11 November 2013 / Accepted: 13 November 2013 / Published: 21 November 2013 Abstract: Primary infection with human cytomegalovirus (HCMV) results in the establishment of a lifelong infection of the host which is aided by the ability of HCMV to undergo a latent infection. One site of HCMV latency in vivo is in haematopoietic progenitor cells, resident in the bone marrow, with genome carriage and reactivation being restricted to the cells of the myeloid lineage. Until recently, HCMV latency has been considered to be relatively quiescent with the virus being maintained essentially as a “ silent partner ” until conditions are met that trigger reactivation. However, advances in techniques to study global changes in gene expression have begun to show that HCMV latency is a highly active process which involves expression of specific latency-associated viral gene products which orchestrate major changes in the latently infected cell. These changes are argued to help maintain latent infection and to modulate the cellular environment to the benefit of latent virus. In this review, we will discuss these new findings and how they impact not only on our understanding of the biology of HCMV latency but also how they could provide tantalising glimpses into mechanisms that could become targets for the clearance of latent HCMV. Keywords: cytomegalovirus; latency; immune evasion; apoptosis; gene expression; cellular signalling 6 1. Introduction Human Cytomegalovirus (HCMV) remains a major cause of disease in a number of patient populations who have compromised immune systems, as well as providing an increasing threat to critically ill immuno-competent patients [1 – 4]. These pathologies associated with opportunistic HCMV infections can be, in part, associated with a key characteristic of the virus: the ability to establish lifelong latent infection of the human host and, crucially, reactivate [2,5]. A wealth of studies from a number of laboratories using naturally latently infected cells has led to an informed consensus that the cells of the myeloid lineage represent at least one important site of HCMV latency, persistence, and reactivation (reviewed in [6]). Thus, at a cellular level, there is a clear and intimate link between myeloid differentiation and natural HCMV reactivation [7 – 14]. Furthermore, the use of experimental infection of non-permissive primary cells and cell lines in vitro are generating snapshots of the complex regulation of HCMV gene expression at a molecular level [15 – 25]. However, these studies have focussed predominantly on the regulation of major immediate early (MIE) gene expression because the critical switch to a reactivating phenotype is dependent on the triggering of MIE gene expression from quiescence. In many cases, the species specificity of HCMV has driven these analyses to be performed in experimental cell culture models and, ultimately, on tissue derived from healthy HCMV seropositive individuals which has then been analysed ex vivo . As a result, the mechanisms that control HCMV latency and persistence in vivo , at an organism level, have relied on the extrapolation of studies performed in vitro or using animal model surrogates such as murine CMV [26]; guinea pig CMV [27] and, more recently, non-human primate CMV strains [28]. Consequently, the inability to perform analogous studies in humans has likely contributed to the perception that HCMV latency is essentially a relatively quiescent infection. However, as techniques for studying HCMV at a molecular level have become increasingly powerful, it is now emerging that latent HCMV infection profoundly modulates the latently infected cell and the surrounding cellular environment. These effects act in concert to maintain latent carriage and this depends on, at least in part, the expression of a subset of virally encoded gene products. In this short review, we will examine our current knowledge of HCMV latency with particular emphasis on recent data which suggest that HCMV imparts a distinctive signature on latently infected cells. These latency-associated changes underpin the successful persistence of this virus in vivo and, importantly, could direct novel therapeutic strategies to target latency and reactivation of this important human pathogen. 2. Background — HCMV Latency and Reactivation Following primary infection, HCMV establishes a latent infection of the CD34+ haematopoietic cell population in the bone marrow [29,30]. The prevailing view is that, ultimately, the major immediate early promoter (MIEP) is profoundly suppressed in these cells [6] and that this is achieved through cellular transcriptional repressors directing histone-modifying enzymes to impart repressive post-translational modifications of MIEP-associated histones [6]. During latency, the chromatin structure of the MIEP bears all the hallmarks of transcriptional repression: tri-methylation of histone H3 (lysine 9 and 27) and recruitment of heterochromatin protein-1 (HP-1) coupled with a 7 concomitant absence of histone acetylation on histone H4 [11,16,17,25]. Consequently, HCMV MIE gene expression, and lytic gene expression in general, is profoundly repressed in CD34+ progenitor cells. This chromatin phenotype is maintained in the monocyte cells derived from these progenitors [11,31] and it is only upon cellular differentiation that robust IE gene expression is observed [7,8,11,12,32]. The detection of IE gene expression in dendritic cells (DCs) is consistent with the histone modifications present at the MIEP in these terminally differentiated myeloid cells [11,31]. For instance, HP-1 is no longer associated with the MIEP — likely due to extensive de-methylation of histones at lysine residue 9 (methylation at this residue being important for HP-1 binding to chromatin [33]) and, in these cells, the MIEP is associated with predominantly acetylated histones. Thus, the presence of repressive or activatory chromatin marks around the MIEP correlates with the expression of viral major IE RNA and the latency/reactivation phenotype of the virus [11,31]. Importantly, and consistent with molecular analyses, infectious HCMV progeny cannot be recovered from myeloid progenitor cells i.e. , CD34+ cells or granulocyte – macrophage progenitors (GMPs) unless they are co-cultured under conditions that promote cellular differentiation or activation [9,11,34]. Analogous models of histone-mediated regulation of viral lytic gene expression also underpin studies of herpes simplex virus and Epstein – Barr virus and thus represent a common unifying theme in the biology of herpesvirus latency and reactivation [35,36]. The molecular model of HCMV latency in the myeloid lineage, derived from analyses of natural latency, has been reviewed extensively elsewhere [6,37,38] and has helped provide an initial understanding of the underlying mechanism for the differentiation-dependent reactivation of HCMV. It is worth noting, however, that other studies using experimental infection models of latency and reactivation have essentially recapitulated the key observations made with natural models of latent infection and this gives confidence that wider studies involving experimentally latent models will have in vivo relevance. 2.1. The Transcriptional Landscape of Latent HCMV HCMV encodes anywhere between 170 and 751 ORFs all of which are believed to be expressed at some stage during lytic infection [39,40]. Furthermore, the virus also encodes a number of microRNAs (miRNAs) which, during lytic infection, have been shown to target and regulate both cell and viral gene expression [41 – 43]. In contrast, the transcriptional landscape in latency is less clear. The earliest studies identified a number of transcripts arising from the MIE region of HCMV but no function was assigned to them [44,45]. Furthermore, deletion of the putative ORFs encoded by these latency-associated transcripts appeared to have little effect on HCMV latency in vitro [46]. As such, it was speculated that HCMV could exist in latency in a relatively quiescent state and that the normal transit and differentiation of latently infected CD34+ cells into the periphery was sufficient to trigger HCMV reactivation. Indeed, transcriptional quiescence during latency would provide the ideal mechanism for evasion of the robust immune responses known to be present in HCMV seropositive individuals [47]. However, a number of aspects of the known biology of HCMV are at odds with the view that HCMV is maintained in a totally quiescent state. For instance, if virus is carried long-term in the myeloid lineage, how is the latent genome maintained in cells which will, at least at some stage of their lifespan, proliferate? Although no latent origin of replication has been definitively identified for HCMV, it has been suggested that a mutation in the MIE region had a 8 carriage defect during latency in GMPs [48] and more recent work has suggested UL84 may act to maintain viral sequences [25]. Furthermore, an overt characteristic of HCMV latency is the carriage of the viral genome in the cells of the myeloid lineage and, particularly, the monocyte lineage [49 – 51] but not lymphocyte or polymorphonuclear cells [50] despite the fact that latent infection is seeded in a pluripotent progenitor cell type [29,30]. Potentially, this could be explained in alternative ways: the virus actively promotes myelopoiesis of infected CD34+ cells or, HCMV may preferentially promote the survival of myeloid committed progenitors or, finally, HCMV cannot combat anti-viral mechanisms in cells committed to the lymphoid lineage. Arguably, all these scenarios suggest an active process involving viral latency-associated functions during latent infection. A number of studies over the last 10 years or so have applied increasingly sensitive techniques to determine whether viral gene expression occurs during latent infection. Two independent microarray analyses identified a number of transcripts expressed during experimental latency [34,52] and, importantly, some have been subsequently confirmed during natural latency; including UL138, UL81-82ast (LUNA), as well as a splice variant of UL111A, which encodes a viral interleukin 10 (vIL-10) termed LAcmvIL-10 [24,53 – 55]. These, and subsequent studies, have also shown that the initial infection of undifferentiated myeloid cells with HCMV to establish experimental latency results in a burst of temporally dysregulated viral transcription from a number of gene loci, including MIE gene expression, at very early times post infection [25,32,34]. However, it remains unclear what this means in the context of latent infection. It is tempting to speculate that this gene expression is important for preparing the cell for latency — akin to that proposed for the establishment of EBV latency [56]. However, there is no evidence, as yet, that cells which initially express lytic antigens go on to establish long-term latency. It is possible that the extremely high MOIs used to establish latent infections in vitro results in a sub-population of lytically or abortively infected cells which are, ultimately, unviable and die, leaving the true latent population. Regardless, what is generally accepted is that HCMV has a very distinct transcriptional profile during latent infection, quite different from lytic infection. The expression of a number of viral genes has now been described during latency and these are summarised in Table 1. For the remainder of this review, we will focus on emerging stories regarding the manipulation of latently infected cells by HCMV and how, in some instances, viral gene products may contribute to this. Table 1. Gene products and functions during latency and lytic infection. Gene Product Latent Function Lytic Function References CLTs Unknown Regulation of anti- viral 2’5’ OAS expression (ORF94) [44 – 46,57] UL138 Regulation of TNFRI (up) and MRP1 (down), repression of the MIEP(?) Regulation of TNFRI (up) and MRP1 (down), virus maturation (133-138 locus) [53,58 – 61] UL81-82ast Promotes UL138 gene expression. Unknown [24,55,62] LAvIL-10 Down-regulation of MHC class II expression, immune evasion Unknown — cmvIL-10 expressed during lytic infection [54,63] Lnc4.9 Binds Polycomb repressor complex 2, Silencing of the MIEP Unknown [25] 9 Table 1. Cont Gene Product Latent Function Lytic Function References UL84 Genome maintenance DNA replication, UTPase activity, transcriptional regulation [25,64 – 67] US28 Unknown GPCR, induces cell signalling and cell migration, agonist of the MIEP [68 – 74] UL144 Unknown TNF superfamily member, hijacks NF-kB signalling, immune evasion? [75 – 78] 3. Mechanisms Targeted during HCMV Latency 3.1. Viral Evasion of Cell Death Pro-death signals in response to infection represent a very significant obstacle for many pathogens. Consequently,