Title

I. Cell-dsRNA interactions and the induction of an antiviral state. II. Quantification and characterization of a chicken serum dsRNA endoribonuclease. III. Formation of viral pseudotypes between retroviruses and dsRNA bearing viruses

Date of Completion

January 2008

Keywords

Biology, Cell|Biology, Virology

Degree

Ph.D.

Abstract

Cells respond to dsRNA, a product of viral replication, with the production of interferon (IFN) and the development of an antiviral state (AVS). AVS is a state in which cells inhibit viral replication and is characterized by the expression of many new proteins. The AVS induction by dsRNA in IFN producing (primary chicken embryo cells; CEC) and non-IFN producing cells (quail; LSCC-H32) were compared, as well as the role of dsRNA (synthetic or viral) extracellular or intracellular interactions in the induction of an AVS. CEC cells were more sensitive to minute amounts of synthetic dsRNA than LSCC-H32. Treatments of LSCC-H32 cells with NH4Cl, an inhibitor of proper endosomal processing, demonstrated that the cytosolic presence of synthetic dsRNA forms was not necessary for the induction of an AVS. Also, NH4Cl treatments demonstrated that endocytosis is important for Avian Reovirus (ARV) to successfully infect a cell. Furthermore, AVS induction was tested in the presence of 2-aminopurine (2-AP), an inhibitor of protein kinase R (PKR), a cytosolic dsRNA binding protein. 2-AP had little effect on AVS induction by synthetic dsRNA [poly r(I)-poly r(C); (pIC)], demonstrating that pIC does not enter into the cytoplasm and it does not react with PKR. ^ Our findings indicate that QM5 cells, a quail cell line, do not enter into an AVS when treated with synthetic forms of dsRNA (pIC polylysine carboxymethylcellulose; pICLC). However, when pICLC is treated with lipofectamine (a cationic surfactant), an AVS is induced in QM5 cells. This indicates that pICLC by itself is not an effective transfection vector for QM5 cells and lipofectamine is needed to assist pICLC to enter the QM5 cells. ^ The chicken dsRNase was quantified during the course of chicken development from embryo to adult and it was found to possess an endoribonucleolytic activity. Finally, the LSCC-H32 cells, which were transformed by avian leukosis virus (ALV) and actively producing virus, when treated with UV-inactivated viruses (ARV) produced a factor, which was able to induce an AVS in CEC. The factor was found in the supernatant medium and was sensitive to: (a) low pH; (b) heat; (c) UV-light; and (d) polyclonal antibodies to the inducing viruses and to ALV. From the above it was concluded that the antiviral factor found in the supernatants of UV-ARV treated LSCC-H32 cells is a viral pseudotype formed between the infecting ARV virus and the endogenous ALV. ^