Interferon Induction by Synthetic Polynucleotides: Importance of Purine N-7 and Strandwise Rearrangement (primary rabbit kidney cells/vesicular stomatitis virus/poly(I) poly(C)/poly(A) poly(U))

The antiviral activity and interferon-in- ducing ability of single-, double-, and triple-stranded poly- nucleotides, modified at pyrimidine C-5 or purine N-7, were evaluated in primary rabbit kidney cells challenged with vesicular stomatitis virus.(l) There is a parallel in- crease in antiviral activity and the temperature at which double-stranded polynucleotides rearrange to inactive triple-stranded complexes. (2) When the purine N-7 of (A)n is replaced by CH, all resulting double-stranded complexes fail to provide antiviral protection or to induce interferon, even though such complexes meet all requirements pre- viously recognized for interferon induction. (3) Competi- tion experiments between inactive and active polynucleo- tides indicate that single-stranded polynucleotides ap- parently do not bind to the cellular receptor sites for inter- feron induction, whereas triple-stranded complexes and inactive double-stranded complexes bind to such receptor sites but, probably for conformational reasons, fail to trig- ger the necessary message for interferon induction. An understanding of the structural parameters governing the induction of interferon by synthetic polynucleotides may be a prerequisite for the design of a clinically useful interferon inducer. The examination of a number of modified poly- nucleotides has led to the delineation of at least three charac- hypothesis; furthermore, the temperature at which (A)n, (U)0 rearranges to (A)n'2(U)n is 10--12 higher than the tempera- ture at which most antiviral assays are performed (Table 1). This would require the ad hoc assumption that the cellular environment produces conditions especially favorable for the rearrangement. We have explored two separate approaches in an attempt to define more clearly the importance of such strandwise (2 -* 3) rearrangements in the interferon system. I. Determination of the Antiviral Activity of M'odified (A)n' (U)n Systems That Differ in T2,(2-3), the Temperature at Which the Double-to-Triple-Strand Rearrangement Occurs. In addition to (A), (U),, we examined the (A),n (rT), and (A)n' (br5U), complexes, since the transitions occurring within these systems are well understood. Introduction of such sub- stituents as bromine or methyl at C-5 of the pyrimidine strand of (I),' (C)0 does not lead to a significant change in antiviral activity (11, 16). II. Evaluation of the Antiviral Activity of a Modified (A)n' (U)n System That Cannot Form a Triple-Stranded Complex under Any Conditions. The (c7A)n. (U)n system fits this de- scription, since it possesses the same hydrogen bonding scheme as (A). (U)n, but, due to the absence of the purine N-7, cannot form a complex equivalent to (A)n 2(U)0 by means of Hoogsteen base pairing. Since the duplex (c7A),. (U)n melts out to single-stranded structures at about 30° in 0.1 M salt (17), we prepared the complexes of (c7A)n with (rT)n and (br5U)n, since such substitutions lead to substantial elevations of Tm in the (A)n (U)n and (I)n ' (C)0 systems (18).