Mode of Action of the Anti-AIDS Compound Poly(I)·Poly(C12U) (Ampligen): Activator of 2′,5′-Oligoadenylate Synthetase and Double-Stranded RNA-Dependent Kinase

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Abstract
The mismatched double-stranded RNA (dsRNA), poly(I) · poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 μg/ml (80 μg/ml) of poly(I) · poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I) · poly(C12U) markedly extends the duration of the transient increase of 2′,5′-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I) · poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I) · poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I) · poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (≥3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I) · poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10−7 to 10−6 grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I) · poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I) · poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.