2-5A-DNA conjugate inhibition of respiratory syncytial virus replication: effects of oligonucleotide structure modifications and RNA target site selection

To define more fully the conditions for 2-5A-antisense inhibition of respir atory syncytial virus (RSV), relationships between 2-5A antisense oligonucl eotide structure and the choice of RNA target sites to inhibition of RSV re plication have been explored. The lead 2-5A-antisense chimera for this stud y was the previously reported NIH8281 that targets the RSV M2 RNA. We have confirmed and extended the earlier study by showing that NIH8281 inhibited RSV strain A2 replication in a variety of antiviral assays, including virus yield reduction assays performed in monkey (EC90 = 0.02 mu M) and human ce lls (EC90 = 1 mu M). This 2-5A-antisense chimera also inhibited other A str ains, B strains and bovine RSV in cytopathic effect inhibition and Neutral Red Assays (EC50 values = 0.1-1.6 mu M). The 2'-O-methylation modification of NIH8281 to increase affinity for the complementary RNA and provide nucle ase resistance, the introduction of phosphothioate groups in the antisense backbone to enhance resistance to exo- and endonucleases, and the addition of cholesterol to the 3'-terminus of the antisense oligonucleotide to incre ase cellular uptake, all resulted in loss of activity. Of the antisense chi meras targeting other RSV mRNAs (NS1, NS2, P, M. G, F, and L), only those c omplementary to L mRNA were inhibitory. These results suggest that lower ab undance mRNAs may be the best targets for 2-5A-antisense; moreover, the act ive 2-5A antisense chimeras in this study may serve as useful guides for th e development of compounds with improved stability, uptake and anti-RSV act ivity. (C) 1999 Elsevier Science B.V. All rights reserved.