Viral infections of the intestine and liver are significant causes of human morbidity and mortality. Understanding the molecular mechanisms of such infections will lead to new approaches in their treatment and control. In addition, basic understanding of the role of cellular genes in viral pathogenesis may provide new targets for therapy of known as well as emerging infectious agents. Our objectives are: (i) to establish mutant cell lines that resist lytic reovirus infection and contain no infectious reovirus, (ii) to determine the DNA sequence of the mutated genes that confers resistance to virus replication, (iii) to confirm that mutated genes truly provide resistance to infection, and (iv) to characterize functional relationships of the cellular genes mutated to the reovirus replication cycle. We will use insertional mutagenesis with retrovirus vectors (U3 gene-traps) to mutate cellular genes and then select mutant cells that resist infection with reovirus. To examine cellular genes associated with reovirus susceptibility we will select cellular mutants obtained by infection of rat intestinal epithelial (RIE-1) cells with a retrovirus vector (U3 gene-trap) that is modified to encode a reporter gene and requires a cellular promotor for its expression. Thus, these vectors """"""""trap"""""""" cellular genes that are transcriptionally active (functional). Selection of mutant cells following U3 gene-trapping containing neomycin phosphotransferase is done in media containing geneticin (G418) followed by reovirus infection. For other experiments, we will utilize U3 gene-trap retroviruses containing beta-galactosidase as a reporter gene (instead of neomycin phosphotransferase) to trap genes that have lower levels of expression. In this case, mutant cells will be assessed for beta-galactosidase (Zlac) activity following reovirus infection to insure insertion of the U3 gene-trap retrovirus has occurred. Cells resistant to reovirus infection are characterized for the gene disrupted by the retrovirus """"""""trap"""""""" event by standard molecular biologic techniques. Once genomic sequence of the disrupted gene is determined, the expression of the gene will be determined in relationship to organ or developmental specificity. The function of isolated genes in the reovirus replicative cycle will be determined by assessing binding of virus, internalization, uncoating, transcription, translation and packaging in the mutant cells. To date, we have accomplished the development of cell lines and clones that are infected with a retrovirus that disrupted only one cellular gene per cellular clone and are rendered resistant to lytic infection by reovirus. These cells resist reovirus infection and contain no infectious virus. Preliminary sequence data of two of the disrupted genes suggests that novel cellular genes will be found.
| Organ, Edward L; Nalbantyan, Christopher D; Nanney, Lillian B et al. (2004) Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication. DNA Cell Biol 23:430-41 |
| Organ, E L; Rubin, D H (1998) Pathogenesis of reovirus gastrointestinal and hepatobiliary disease. Curr Top Microbiol Immunol 233:67-83 |
