Thomas L. Benjamin, Ph.D - US grants

Attempts will be made to regulate the expression of the c-src and other cellular proto-oncogenes. The approach will involve construction of plasmids that encode proto-oncogene antisense RNAs that can act to arrest the translation of the homologous mRNA in vivo. Such plasmids will be transfected into polyoma transformed rat and mouse fibroblasts, as well as normal primary and established cells, and teratocarcinoma cells. A long term objective includes the insertion of these plasmids into mouse embryos and establishment of transgenic mice.

The aim of the proposed research is to further our understanding of the mechanism of neoplastic cell transformation by polyoma virus at the molecular biological level. Advantage will be taken of our current knowledge of the genetics of the virus as it affects the transformation process, of DNA sequence data in the HR-T region of the viral genome the expression of which is known to be essential for transformation, and of RNA splicing events which occur in sequences encoded in this region of the viral DNA. We will pursue these aims by applying a specific in vitro site-directed mutagenesis procedure using synthetic single-stranded oligonucleotide DNA sequences. Sequences 10-12 nucleotides long and chosen to have a specific base mismatch with the viral DNA will be annealed to single-stranded circular viral DNA, and extended with DNA polymerase in vitro. These heteroduplexes will then be used to infect cells, and the resulting virus lysates will be used to obtain multiple single plaque isolates. Lysates grown from the latter will then be screened for the desired phenotype of T antigen analyses.

This proposal is to continue studies to characterize the hr-t gene of polyoma virus and its role in virus growth and malignant cell transformation. We shall seek to characterize the small and middle tumor antigens which constitute the dual products of the hr-t gene. Specific aims include: 1), mapping of sites of in vivo phosphorylation of mT antigens by cellular kinases, and the potential role these phosphorylations have in activating the mT-associated tyrosine-specific kinase activity; 2), use of synthetic peptide antigens to raise antisera capable of reacting specifically with mT protein; 3), use of such antisera to affinity purify mT proteins; 4), determine whether the tyrosine kinase activity is intrinsic to mT or resides in a cellular enzyme; 5), studies of cellular phosphoprotein metabolism and how its affected by hr-t viral gene, using an anti-phosphotyosine antiserum and other approaches; 6), studies on the role of the hr-t gene in virus maturation, possibly at the level of capsid protein phosphorylation; 7) studies directed toward sT antigen, its isolation and purification, and functional properties, and, 8), studies directed toward the 36K and 63K "non-viral" tumor antigens, their associations with viral proteins, and possible functional significance.

This application proposes to merge the work presently supported by three separate grants dealing with various aspects of work on polyoma virus. Molecular biological studies of polyoma virus interactions with susceptible cells in culture are being pursued with the particular goal of understanding the roles of the middle T and small T proteins in cell transformation and in productive viral infection. Structure-function studies of middle T using oligonucleotide mutagenesis are proposed, with the aim of elucidating the interaction of this viral protein with cellular protein kinases, and possibly with phospholipid metabolism. With respect to the virus growth cycle, similar approaches are being used to study VP/1 and its phosphorylation sites in order to gain a better understanding of how such modifications of the major capsid protein may be important in assembly of the virus particle. A third area deals with interactions of the virus with the intact host, in particular with the immune system and recognition of the polyoma-specific transplantation antigens.

DESCRIPTION: The goals of this project are to define the pathways of induction of tumors in the mouse by polyoma virus at the molecular level. To this end, mutant strains of virus that have been well characterized in cell culture systems will be inoculated into newborn mice; the latter will then be followed for development of the various tumor types which the wild-type virus is known to induce. Site- directed mutagenesis will be used to modify particular regions of the middle T viral oncogene. These mutants will then be characterized biochemically and in cell transformation assays in order to understand more fully the interactions of middle T with cellular enzymes, including the tyrosine protein kinase pp60c-src, phosphatidylinositol 3-kinase and protein phosphatase 2A. Analogues of inositol modified at the 3-position will be examined as potential inhibitors of 3-phosphoinositide metabolism in vivo. Mutants of large T, or other viral proteins potentially involved in interactions with host tumor suppressor genes, will be sought and characterized. Cellular kinases that phosphorylate the major viral capsid protein VP1 will be identified. Attempts will be made to identify additional cellular elements involved in a cascade between middle T and VP1; this cascade is viewed as overlapping with a mitogenic pathway of the host cell. Finally, an attempt will be made to identify, map, and ultimately clone, genes of the host that confer susceptibility or resistance to tumor induction by the virus.

[unreadable] DESCRIPTION (provided by applicant): The long-term objectives of this application are to understand the mechanisms of cell entry by the mouse polyoma virus. This virus has a broad host range. It replicates and induces tumors in a wide variety of cell types in its natural host. The Specific Aims are first to determine whether certain glycolipids constitute the only class of cell receptors for the virus or whether certain glycoproteins may also serve as functional receptors. This will be done using a mouse deficient in glycolipid biosynthesis but unaffected in glycoprotein pathways. Investigations will be carried out to identify the steps in virus disassembly in the endoplasmic reticulum and the cellular factors the virus utilizes to undergo the necessary steps of disassembly and to enter the nucleus. Factors under investigation in this regard are the oxidoreductase ERp29, protein disulfide isomerase, the derlin chaperones, and Ran GTPase. A specific goal is to generate in vitro and characterize the altered particle that is primed for membrane insertion and escape from the ER. Finally, in collaboration with Dr. Xiaowei Zhuang's lab at Harvard, attempts will be made to follow single virus particles in live cells as they enter and establish infection. We will attempt to produce polyoma particles labeled both on the minichromosome and the outer capsid protein with fluorescent probes to study decapsidation and separation of these components. The processes of cell entry by many viruses are not fully understood. The polyoma group includes several viruses that are pathogenic in humans, including JC, BK and possibly SV40. Understanding the pathways of cell entry by these viruses may suggest opportunities for anti-viral therapy or prevention. [unreadable] [unreadable] [unreadable]

DESCRIPTION: (Adapted from the Investigator's abstract): We propose to use the polyoma virus-mouse system to investigate aspects of virus-host interactions leading to induction of tumors. Two areas of general relevance to the biology of cancer will be investigated. The first concerns the apparent inability of polyoma virus to block the actions of p53, raising questions of the role of genomic instability in driving tumor development in this system. This will be approached using molecular cytogenetic techniques to study genomic changes in polyoma-induced mouse tumors, studies in tissue culture to determine how the virus may override or bypass p53, and derivation of transgenic mice expressing regulatable polyoma T (tumor) antigens. The second area concerns the roles of the host genetic background in determining patterns of susceptibility and resistance to tumors. Three mouse strains exhibiting different tumor responses will be studied - 1) susceptibility to tumor induction based on failure to develop tumor immunity, 2) tissue specific resistance to mammary tumors, and 3) propensity of bone tumors to metastasize to the lung. Genetic and physiological approaches will be used in attempts to understand the bases of these host phenotypes.

DESCRIPTION (provided by the applicant): This project concerns the use of a 'tumor host range' (T-HR) selection procedure for isolation of host range mutants of Polyoma virus. The procedure involves screening and selection of virus mutants for their ability to grow on non-polyoma transformed or tumor-derived cells and inability to grow in normal primary mouse cells. The procedure is coupled to the yeast two hybrid system using wild type virus T antigen sequences as bait to screen mouse embryo cDNA libraries. Absence of interaction of target cDNAs with the mutant bait is taken as an indication of a physiologically relevant target in virus replication or transformation. The procedure is viewed as a possible way to uncover new tumor suppressor genes or other factors with which the virus must interact in normal cells in order to grow and which are altered or missing in cancer cells. We will use results from a partially characterized T-HR mutant and its putative tumor suppressor gene target to produce a mouse model of ovarian carcinoma.