Phosphorothioate oligonucleotides inhibit human immunodeficiency virus type 1 fusion by blocking gp41 core formation

Abstract

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2'-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (>or=30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.

FIG. 1.

Nucleic acid chemistry used in the experiments. Phosphorothioation (which stabilizes and increases hydrophobicity to ONs) and 2′-O-methylation (which only stabilizes ONs) were used to alter the chemical properties of Randomers used in this study.

FIG. 2.

Synthesis and stability of modified ONs. (A) Size analogues of Randomer 1, 2, and 3 ONs were resolved on 10% urea-acrylamide gels to verify their size. In all cases, >90% of the oligo was observed to be full length. Size standards (in nucleotides [nt]) are identified on the right. (B) The temporal stabilities of 40-mer ONs with Randomer 1, 2, or 3 chemistries in fetal calf serum at 37°C were compared to the unmodified 40-mer degenerate ON. Following incubation for various times, ONs were resolved as for panel A. Degradation in both the 40-mer Randomer 1 and 2 ONs was undetectable after 96 h, the 40-mer Randomer 3 ON showed partial degradation beginning at 48 h, and the unmodified 40-mer degenerate ON was almost completely degraded by 48 h. ONs dissolved in PBS are provided as controls on the left.

FIG. 3.

(A) Effect of time of addition on the antiviral activity of a 40-base Randomer 1. Various control compounds (see Results) and Randomer 1 were added to cultured cells at various times during (<2 h) or after (>2 h) infection with HIV-1. All compounds were used at IC₉₉. (B) Inhibitory activity of PS-ONs in attachment and fusion. A 40-base Randomer 1 PS-ON was tested in an attachment-fusion assay against both X4- and R5-tropic HIV-1 strains.

FIG. 4.

PS-ON size and phosphorothioation dependence on inhibition of 6-HB formation by FN-PAGE. (A) Different-size analogues of Randomer 1, 2, and 3 ONs (200 μM) were added to the N36 peptide (40 μM) before the addition of the FITC-C34 peptide (40 μM). A size-dependent inhibition was observed with Randomer 1 and 2 PS-ONs, but no significant inhibition was observed with Randomer 3 ONs of any size. (B) Dose response of the inhibition of the N36/FITC-C34 complex (both at 40 μM) by increasing concentrations of 40-base Randomer 2 or 3 ONs. For simplicity only the region of the gel displaying the 6-HB complex is shown in panels A and B.

FIG. 5.

FN-PAGE analysis of inhibition of 6-HB formation by PS-ONs. The 40-base Randomer 1 or 2 ONs (200 μM) were added to the N36 peptide (40 μM) before or after the addition of FITC-labeled C34 peptide (40 μM). The N36/FITC-C34 6-HB complex has a retarded mobility (as indicated on the right of the gel). Randomers 1 and 2 were active in preventing 6-HB formation when they were incubated with N36 prior to the addition of FITC-C34.