INVESTIGATING THE ROLE OF THE ADENOVIRUS TYPE 5 EARLY REGION 1B 55 KDA PROTEIN IN VIRAL REPLICATION IN NORMAL HUMAN CELLS

Abstract

The adenovirus family includes ubiquitous human pathogens that naturally cause mild self-limiting to severe respiratory tract infections. The subject of study for nearly 60 years, many fundamental aspects of molecular biology were elucidating in adenoviral experimental systems. In recent years adenovirus has emerged as a popular vector in the development of oncolytic and gene therapies, and for vaccine delivery. Despite an extensive history of research and great interest in adenoviruses as tools in medical and industrial biotechnology applications, fundamental aspects of the virus' biology remain poorly understood. In particular, understanding the functions of the early gene region 1B 55 kilodalton protein product, E1B 55 kDa, continues to challenge the field. This multi-functional protein mediates effects ranging from the transformation of cells via the inhibition of apoptosis, to the modulation of nuclear mRNA export, to the protection of viral DNA synthesis, depending on the host cell species and tissue of origin, and genetic makeup.

Two major avenues of investigation are described in this thesis. Our primary interest lay in the elucidation of the role of E1B 55 kDa in supporting viral replication specifically in the context of infection in primary, untransformed human cells. These better reflect the host environment during natural adenovirus infection than immortalized and transformed cell lines typically used in studies of virus biology. Findings over the course of these investigations led to the discovery of a new role played by E1B 55 kDa in blocking the inhibition of viral DNA synthesis by the type I interferon response (Chapter 2), and resolved conflicting reports in the field regarding the dependence of viral DNA synthesis upon the presence of the E1B 55 kDa protein (Chapter 3). The second avenue of investigation arose from the chance discovery of an infectivity defect attributable to a substitution mutation in the coding sequence of the viral terminal protein (TP, Chapter 4). Plaque-forming units (pfus) of the E1B 55 kDa-null virus, host range 6 (Hr6), commonly exploited in studies of E1B 55 kDa function, were observed to contain an abundance of noninfectious genomes in experiments analyzing viral DNA replication, which could not be attributed to the mutations in the E1B region. A proteolytic processing defect in TP was characterized in this virus strain that led to rapid degradation of the majority of viral genomes that entered the cell, representing a novel infectivity phenotype. Finally, ongoing experiments aimed at identifying E1B 55 kDa interacting partners that are novel and/or involved in mediating the interferon-inhibitory effects reported in Chapter 2 are described briefly (Chapter 5). These preliminary results demonstrate the feasibility of a novel proteomic approach to identifying E1B 55 kDa-binding proteins, which represents an important tool for the future elucidation of mechanisms of E1B 55 kDa action.