Role of the Human Cytomegalovirus Major Immediate-Early 1 Protein in Viral Reactivation from Latency

Human cytomegalovirus or hCMV is one of eight herpesviruses that are known to infect people. This virus is very common, infecting one out of two persons in the UK. Once hCMV is in a person's body it stays there for life, usually in a dormant state referred to as "latency". In latency, few viral genes are active and no virus is produced. Although hCMV likely remains latent in several places of the human body, blood and bone marrow cells of the "myeloid" type are the best known sites of hCMV latency. Most healthy adults and children who become infected will have few signs or symptoms and no long-term effects from hCMV. However, people at risk include those with weakened immune systems including individuals who receive transplants, undergo cancer chemotherapy or are positive for HIV. It is well established that in these people, hCMV can cause life-threatening disease affecting multiple organs. However, the fact that hCMV is also the leading cause of congenital (present at birth) infections worldwide is less appreciated, and so is the socioeconomic impact of this virus as the most common cause of childhood hearing loss and brain disorders. Two to three babies are damaged by hCMV every day (almost 1,000 babies every year) in the UK. In fact, hCMV causes more birth defects and childhood deaths than Down's syndrome, toxoplasmosis or listeriosis and is a greater global health concern than the Zika virus. Yet, congenital hCMV lacks awareness among health care workers and the public. Furthermore, no vaccine for this virus is available and no approved treatment for congenital infection exists, since all available drugs have dangerous side effects. Consequently, there is an urgent need to identify new molecular targets and better drugs for hCMV.

Although hCMV may cause serious symptoms upon initial infection of people at risk, disease more typically ensues when the virus "reactivates" in latently infected individuals. Reactivation from latency is considered to be a stepwise process that ultimately results in the global activation of viral genes and production of infectious virus. How hCMV reactivates from latency is far from clear, especially at the molecular level. Much of our previous research has centred on an hCMV protein known as immediate-early 1 or IE1. IE1 is believed to be the very first viral protein produced during hCMV reactivation and is therefore expected to have an important role in this process. Based on preliminary experiments, we propose that IE1 serves as a key viral factor in hCMV reactivation via at least two molecular mechanisms involving host proteins of the signal transducer and activator of transcription (STAT) family and nucleosomes, the basic building blocks of chromatin, respectively. We will employ state-of-the-art technology using infected human myeloid cells as a model to elucidate how exactly IE1 modulates molecular pathways linked to STAT proteins to reactivate virus production. Likewise, we will identify chromatin-based "epigenetic" mechanisms underlying IE1-mediated reactivation of viral genes. Finally, we will evaluate agents targeting the molecular events linked to IE1-mediated reactivation for their antiviral capacity and potential to serve as candidates for drug development. The results from this work will considerably improve our limited molecular understanding of how hCMV reactivates from latency to cause disease. They will also reveal new strategies to combat this important, but somewhat neglected, human pathogen.

Human cytomegalovirus (hCMV) is the leading cause of birth defects and a major pathogen in immunocompromised patients, yet prevention and treatment options are limited. Following primary infection, hCMV establishes lifelong latent infection in its host. Although much of the disease burden associated with hCMV infection has been linked to reactivation of latent virus, the molecular mechanisms underlying this process have remained unclear. The proposed project will investigate key molecular events required for hCMV reactivation in myeloid cells focussing on the viral major immediate-early 1 protein (IE1). IE1 is among the very first proteins expressed during hCMV reactivation and therefore an obvious candidate for regulating this process. The function of IE1 has been extensively studied in fibroblasts permissive for viral replication. However, how IE1 may contribute to hCMV reactivation from latently infected cells has not been explored. Our previous work in fibroblasts has shown that IE1 targets proteins of the signal transducer and activator of transcription (STAT) family to modulate central cytokine-induced signalling pathways including interferon responses. We have also shown that IE1 docks with the nucleosome surface. We propose that the IE1-STAT and IE1-nucleosome interactions are individually required for efficient hCMV reactivation from myeloid cells. We will elucidate exactly how IE1 may promote hCMV reactivation by rewiring signalling linked to STAT1, STAT2 and STAT3. We will also study how the IE1-nucleosome interaction may impact chromatin composition and function pertinent to viral reactivation including core histone ubiquitylation. Finally, we will explore how the relevant IE1-host interactions may be exploited for novel strategies targeting hCMV reactivation. The results from this study will add considerably to our limited molecular understanding of hCMV reactivation from latency and will identify novel strategies to combat this important virus.

Besides providing short-term impact on researchers and students (1), we anticipate the project to impart opportunities for drug development (2), economic impact (3) and health-related societal impact (4) in the long term.

(1) The staff and students involved in the project will develop professional skills transferable to other academic or commercial sectors. The proposal involves the development of improved biomolecular techniques including iPOND, a method that was previously established in other cellular and viral systems and will be adopted to hCMV chromatin. In addition, the herpesvirus field will benefit from important new information regarding the mechanism of hCMV reactivation from latency, which is a poorly understood process. STAT pathways have become a paradigm for membrane-to-nucleus signalling, they are involved in human autoimmune diseases and malignancies, and their impact is now felt beyond immunology in a number of emerging fields including stem cell biology and metabolomics. Likewise, histone-based epigenetic processes are linked to human disease including cancer. Thus, our research is expected to impact on a broad scientific audience.

(2) There is no preventive vaccine for hCMV and antiviral therapy is limited to a small set of drugs. Although these agents have proven efficacious in the prophylaxis and treatment of hCMV disease, they are linked to significant problems such as toxic side effects. Moreover, all currently approved systemic treatments for hCMV and other herpesvirus infections target a single molecular process, viral DNA replication, and this is linked to viral resistance. Consequently, there is a need for new molecular targets and drugs with fewer and less severe side effects. Notably, the estimated market value of an improved hCMV drug or vaccine amounts to over 1 billion US dollars. The proposed project will explore how the IE1-STAT interactions relevant to hCMV reactivation may be selectively targeted using drug-like small molecules and other agents. STAT signalling has become a prototype for transition from bench to bedside, culminating in the development and clinical implementation of pathway-specific therapeutics. Moreover, cellular drug targets have not been exploited for herpesvirus therapy and less likely lead to resistance compared to viral targets. Thus, our findings will point to new directions in drug development for hCMV.

(3) Opportunities for commercial exploitation of project results (e.g., molecular tools or candidate drugs) will be pursued through the Knowledge Transfer Centre at St Andrews, as outlined in Pathways to Impact. Congenital hCMV infection causes a high burden of disease and, since it affects the very young, it results in long-term morbidity. In the 1990s, the estimated costs associated with congenital hCMV disease for the US health care system amounted to at least 1.86 billion dollars annually, with more than 300,000 dollars per child. The costs of treating hCMV disease in solid organ or stem cell transplant recipients range between 25,000 and over 70,000 US dollars per patient. Although beyond the proximate scope of the proposed project, any improvements in the control of hCMV disease our work may contribute to in the long term could have considerable economic benefits.

(4) The potential impact of research into hCMV reactivation on people affected by this virus, including mothers and critically ill patients, is huge. As highlighted throughout this application, hCMV is a very real but somewhat neglected public health concern, and much of the morbidity and mortality linked to hCMV results from reactivating infections. Our short-term goal is to contribute to a better molecular understanding of how hCMV reactivates from latent infection. An improved insight into these processes will inform the development of new ways for preventing or treating disease caused by this virus with substantial long-term potential benefits for public health and well-being.