Investigations of the mode of action of interferon. Part I: The effects of the copper chelators diethyldithiocarbamate and bathocuproine sulfonate on interferon and its antiviral state. Part II: Studies on the fate of the mengovirus genome in interferon-treated cells

Jay Gregory Calvert, Purdue University

Abstract

Part I. Two structurally unrelated chelators of copper ions, diethyldithiocarbamate (DDC) and bathocuproine sulfonate (BCS), were found to inhibit interferon action. DDC was cytotoxic and inhibited mengovirus growth. In contrast, BCS blocked interferon action without these undesirable effects. The compounds inhibited interferon action by different mechanisms. DDC appears to block interferon action by inducing a generalized inhibition of cellular RNA and protein synthesis, possibly combined with the inactivation of one or more specific enzymatic activities involved in the antiviral state. BCS does not readily penetrate biological membranes. BCS combined with trace copper ions in the growth medium, and the resulting complex bound to all interferons tested, thereby preventing them from interacting properly with cellular receptors. Furthermore, some of the interferons were irreversibly inactivated, probably due to oxidation of cysteine, tyrosine, or tryptophan residues by the BCS$\sb2$Cu(II) complex. BCS was at least as effective as anti-interferon antibody at neutralizing cell-bound interferon, and has a number of advantages over it. Part II. The fate of the parental RNA genome of mengovirus was investigated. The virus was labeled in vivo with $\sp3$H-uridine, purified, and used to infect interferon-treated and control cells. RNA was extracted from these infected cells and analyzed by gel electrophoresis and fluorography. A large fraction of the intracellular label was found in a band of genome length which persisted throughout the course of infection under all conditions. This was interpreted as intracellular virions which failed to uncoat. Light-sensitive, $\sp3$H-labeled mengovirus was then prepared by growing the virus in the presence of neutral red. When cells infected with this virus were illuminated, the labeled RNA in intact virions was selectively removed from the sample during phenol extraction. It is suggested that this is the result of covalent cross-linking of the RNA to viral proteins. Removal of this labeled RNA did not, however, reveal an underlying band of light-resistant RNA. Failure to detect this band is attributed to a lack of sensitivity in the fluorographic detection of $\sp3$H and/or low levels of RNase-mediated degradation during RNA isolation. Methods of circumventing these problems are suggested.

Degree

Ph.D.

Advisors

Simon, Purdue University.

Subject Area

Microbiology

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