Human cytomegalovirus (CMV) is a persistent and latent beta-herpes virus that is carried by over 70% of the population. CMV is usually asymptomatic in healthy people. However, in immunocompromised individuals including newborns, transplant recipients, and AIDS patients, CMV proliferation leads to increased morbidity and/or mortality of these hosts. The high CMV viral load in immunocompromised individuals can induce CMV-associated diseases such as vascular neurological disorders, multifaceted frailty syndrome, pneumonia, colitis, and hepatitis. High-risk hosts for CMV disease include CMV seronegative pregnant women and transplant recipients with a primary CMV infection. Remarkably, CMV is the leading cause of birth defects affecting up to 2.5% of newborns worldwide. Other populations at risk for CMV disease include AIDS patients, patients with cardiovascular diseases, autoimmune disease patients, and the elderly. Moreover, the combination of immunosenescence and high CMV prevalence in the elderly (80-90%) may limit the protective effect of general vaccines in elderly people and influence age-related diseases (e.g. atherosclerosis, rheumatoid arthritis, and Alzheimer’s disease). Despite the protective immunity gained in seropositive people, infection by diverse CMV genotypes and viral reactivation in a compromised immune system can also lead to viral-induced disease and increase in morbidity and mortality. CMV is a major medical problem projected to cost a total of $4.4 billion/year and $300,000/child. Thus, the understanding of the CMV life cycle and its ability to manipulate cellular process for replication and immune evasion is essential to the development of effective and safe anti-CMV therapeutics.

Project 1: Discovery of small compounds as CMV anti-virals

The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we generated CMV variants that express fluorescence proteins or luciferase to be utilized in a high-throughput assay to identify inhibitors targeting CMV entry and the viral life cycle.

Project 2: Identification of anti-CMV biologics

Cytomegalovirus infection is a complex process that requires viral and cellular factors to successfully generate viral progeny. The CMV virion contains glycoproteins complexes consisting of gB, gH/gL/gO (trimer), gH/gL/UL128/UL130/131 (pentamer), and gM/gN that are necessary for entry into fibroblasts, epithelial cells, and endothelial cells. The viral glycoproteins and its complexes interact with cellular factors that allow entry into the cell. One project in the laboratory is to generate neutralizing monoclonal antibodies against human cytomegalovirus that limit viral entry. These reagents are potential therapeutics against CMV and are excellent tools to delineate CMV entry and spread.

Project 3: Cytomegalovirus latency

The ability of human cytomegalovirus to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. We have established a novel short-term in vitro latency model that mimics the events of HCMV latency and reactivation of peripheral blood monocytes in order to study the immunological consequence of latent virus carriage. Infection of human CD14+ peripheral blood monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Our data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, that may play a role in the establishment, maintenance, and reactivation from latency. The modulation of innate immune response is a likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host. Our goal is to define the critical cellular and viral factors involved in the establishment and maintenance of a latent state.

Project 4: Mechanism of viral-induce MHC class I down regulation

ER quality control is an essential biological process involved in multiple facets of cell viability through the recognition of misfolded ER proteins as well as the ability to respond to fluctuations of lipid and metabolic factors. Strikingly, the process of ER quality control and ER-associated degradation (ERAD) processes have been co-opted by pathogens such as viruses to limit immune detection and remain persistent within the host. Human cytomegalovirus is on such virus that has co-opted the ER quality control process to eliminate the immunological protein major histocompatibility complex (MHC) class I molecules; thus preventing the recognition of cytotoxic T cells of HCMV infected cells. MHC class I and misfolded ER proteins are disposed through a four step process: 1) Recognition, the ER degradation substrate is recognizes by cellular factor such as chaperones; 2) Recruitment, the substrate is recruited to the extraction site through ER and cytosolic proteins, possibly through ubiquitin attachment; 3) Dislocation, the targeted protein is extracted through the ER membrane by a process referred to as dislocation likely through a membranous pore known as the dislocon, and 4) Degradation, the substrate is finally degraded in a proteasome-dependent manner. This highly regulated process requires numerous cellular factors that contact the substrate itself and/or a post-translational modification (e.g. ubiquitin) of the substrate to efficiently dispose of the ER substrate. Despite the discovery of many proteins involved in all four steps of the ERAD process, essential factors that regulatory important steps of the processes has not been fully elucidated. We are utilizing the well established viral degradation model system of HCMV US2- and US11-mediated class I destruction to identify important cellular factors that mediate the extraction of class I heavy chains across the ER membrane. The characterization of class I destruction by US2 and US11 continues to be instrumental in delineating the cellular process of ERAD.