Activation of ribonuclease L by (2'-5')(A)4-poly(L)-lysine) conjugates in intact cells

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Abstract
Molecular hybrids were synthesized by coupling (2''-5'')(A)n oligoadenylates or 2-5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2''-5'')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The .alpha.-glycol group of individual (2''-5'')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14,000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5'' end moiety of the (2''-5'')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2''-5'')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2''-5'')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2''-5'')(A)n as in some IFN-treated virus-infected cells. Indeed, (2''-5'')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2''-5'')(A)n in a cell-free radiobinding assay. Moreover, (2''-5'')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells. Poly(L-lysine) thus represents an effective vector for the delivery of (2''-5'')(A)n or its analogues to intact cells and is thus a potentially interesting tool for the study of the biological role of (2''-5'')(A)n and its possible utilization in antiviral and antineoplastic chemotherapy.

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