William S.M. Wold, Ph.D.
William S.M. Wold, Ph.D.
1. Human adenoviruses commonly cause acute infections of the respiratory and GI tracts, the eye, and various other tissues. They also form long-term persistent infections, and their reemergence is a problem in immunocompromised patients. Recently, there has been a great deal of interest in using adenoviruses as vectors for specialized vaccines and for gene therapy.
Aside from their interest as agents of disease, adenoviruses are one of the premier model systems to study the molecular and cellular biology of human cells. Adenoviruses are easy to work with, they are very well characterized, and they pose no risk to the researcher. They are classified as "DNA tumor viruses" because they have a DNA genome, they transform cultured cells to a malignant state, and they can induce tumors in experimental animals (but not in humans). Thus, adenoviruses are excellent models to understand cancer. Adenovirus genes are expressed in the cell nucleus using primarily host cell mechanisms, and many of the adenovirus gene products usurp key regulatory aspects of the cell and convert the cell into a factory for efficient virus replication. Many fundamental discoveries have been made using adenoviruses, including pre-mRNA splicing (Nobel prize awarded in 1993) and identification of novel transcription factors and cellular proteins that control the cell cycle and are the key regulators of malignancy. There is a long tradition of adenovirus research at SLU dating back to the 1950's, and it can be argued that SLU is the birthplace of adenovirus molecular virology.
2. Adenovirus proteins counteract the host immune system. In recent years, using modern techniques in molecular and cellular biology, our laboratory has participated in the discovery of a new class of adenovirus proteins that allows the virus to counteract immunosurveillance. The genes for these proteins are located in the "E3 Transcription Unit" in the adenovirus genome. Over the past two decades, we have conducted many studies on this group of genes. We have determined the DNA sequence of the genes, characterized the multiple overlapping alternatively spliced mRNAs, showed that the seven predicted proteins are in fact synthesized, and discovered the functions for many of the proteins. The seven E3 proteins are depicted as colored bars in the accompanying figure and the functions associated with the proteins are indicated. These functions will be discussed below.
Adenoviruses and the human hosts they infect are involved in an evolutionary battle. As virions are formed in the cell, inflammatory cytokines (intercellular messenger molecules) such as tumor necrosis factor (TNF) are synthesized by localized activated leukocytes. TNF is the prototype for a number of "death ligands" that are members of the TNF family and are expressed on the surface of activated leukocytes. These other ligands are named TNF-related Apoptosis-Inducing Ligand (TRAIL) and Fas Ligand (FasL). These ligands have evolved to mediate destruction of adenovirus-infected cells by killer cells of the immune system (macrophages, natural killer cells, cytotoxic T lymphocytes). The ligands function by interacting with specific cognate receptors on the surface of infected target cells. The receptors are named TNF Receptor 1, TRAIL Receptor 1, TRAIL Receptor 2, and Fas. When engaged by TNF, TRAIL, or Fas, respectively, these receptors trimerize on the cell surface and induce a series of intracellular protein-protein interactions that eventually lead to apoptosis (programmed cell death) of the infected cell. The apoptotic process involves the activation of a specific family of proteases named caspases. TNF is known to inhibit the replication of many viruses and to kill cells infected by certain viruses.
As mentioned, adenovirus has evolved several proteins that inhibit apoptosis induced by these death ligands. As a result, these adenovirus proteins protect infected cells from elimination by the immune system. These proteins are named RID (Receptor Internalization and Degradation), E3-14.7K, and E1B-19K, and we are attempting to understand how they function. Current work indicates that RID forces the death ligand receptors (TNFR1, TRAIL-R1, TRAIL-R2, Fas) from the cell surface into endosomes which are transported to lysosomes where the receptors are degraded (see the figure). Downregulation of TRAIL-R2 requires the E3-6.7K protein in addition to RID. RID appears to act via a tyrosine-based intracellar protein sorting signal located in the RID protein. The mechanism by which E3-14.7K functions is unknown. Other laboratories have shown that it interacts with a protein involved in TNF signal transduction, although the consequences of this induction are unclear. Both RID and E3-14.7K inhibit the signal transduction pathway that is used by TNF to induce synthesis of arachidonic acid and its metabolites, the leukotriennes and prostaglandins. The leukotriennes and prostaglandins are potent mediators of inflammation; thus, RID and E3-14.7K also probably inhibit the generalized inflammatory response to virus infection. In accord with this prediction, both RID and E3-14.7K inhibit inflammation and pathology in animal models. Other laboratories have shown that the E1B-19K protein is a functional analog of the cellular protein named BCL-2, a well-known protein that inhibits apoptosis.
These various adenovirus proteins represent powerful tools to unravel the complexities of virus infections. By understanding what these proteins do and how they do it, we will learn a great deal about viral pathogenesis, the host's antiviral defenses, immunology, signal transduction, and targeting of proteins to specific cellular organelles
3. Adenovirus Death Protein (ADP) - a novel mechanism for adenovirus spread, and a therapeutic protein in cancer gene therapy. At the very late stages of infection, when the cellular nucleus is packed full of virions, adenovirus synthesizes a unique protein, termed Adenovirus Death Protein (ADP), which promotes cell lysis and thereby allows adenovirus to be released from cells and infect other cells. We are attempting to understand how ADP functions. ADP is an abundant integral membrane glycoprotein that localizes to the inner and outer nuclear membrane and the Goldi apparatus. It has specific domains that allow it to be targeted to membranes and to induce cell lysis.
We are developing adenovirus vectors for cancer gene therapy (see the figure). These vectors are "smart bomb" viruses that replicate in tumors but not normal cells, and they destroy cancerous cells as a natural aspect of virus replication. All the vectors overexpress ADP, and as such they are more cytolytic than ordinary adenoviruses. All the vectors also lack the E3 immunoregulatory genes discussed earlier, and as such they are safer than typical adenoviruses. Some of these vectors are targeted to tumors by a mutation in an adenovirus gene that precludes virus replication in non-cancerous cells. Some of our cancer gene therapy vectors are targeted to tumors by replacing one of the adenovirus transcription control elements (a promoter) with a tissue-specific and/or cancer-specific promoter. Thus, the vector can only replicate in cancerous cells of specific tissues. We hope to begin to test these vectors in human cancer trials.