Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro

Abstract

Nucleic acid polymers (NAPs) utilize the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) to target protein interactions involved in viral replication. NAPs are broadly active against a diverse range of enveloped viruses that use type I entry mechanisms. The antiviral activity of NAPs against hepatitis B virus (HBV) infection was assessed in vitro in duck hepatitis B virus (DHBV)-infected primary duck hepatocytes (PDH). NAPs efficiently entered PDH in the absence of any transfection agent and displayed antiviral activity at concentrations of 0.01 to 10 μM, measured by their ability to prevent the intracellular accumulation of DHBV surface antigen, which was independent of their nucleotide sequence and was specifically dependent on phosphorothioation. Higher levels of antiviral activity were observed with NAPs 40 nucleotides in length or longer. The fully degenerate NAP (REP 2006) was active during DHBV infection or when added 12 h after infection. In contrast, an acidic-pH-sensitive NAP (REP 2031) that was broadly active against other viruses displayed antiviral activity when present during DHBV infection but no activity when added 12 h after infection, suggesting that NAPs exert their postentry effect in an acidic environment unique to DHBV infection. Both REP 2006 and REP 2031 displayed negligible cytotoxicity in PDH at concentrations of up to 10 μM, as assessed using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cytotoxicity assay. The antiviral activity of NAPs against DHBV in vitro was strictly dependent on their amphipathic character, suggesting that NAPs interact with amphipathic target(s) that are important for DHBV entry and postentry mechanisms required for infection.

Figures

Fig 1

Structures of various NAPs tested for antiviral activity against DHBV infection in PDH and the role of the chemistry of NAPs in eliciting the antiviral activity. (A) Degenerate NAPs tested for antiviral activity. (B) NAP analogs with a defined chemistry (REP 2117 and REP 2118) or with a defined poly(C) sequence (REP 2031) tested for antiviral activity.

Fig 2

Intracellular localization of Cy3-labeled REP 2006 by PDH. PDH were treated with 1.0 μM Cy3-labeled REP 2006 and then fixed on days 1 (A), 4 (B), and 7 (C) with EAA, as described in Materials and Methods. Cy3-labeled REP 2006, visualized by confocal fluorescence microscopy (emission at 585 nm), was distributed diffusely in the cytoplasm and nuclei of the PDH.

Fig 3

Detection of DHBsAg-positive PDH infected with 250 virus genome equivalents of DHBV per cell and simultaneously treated with NAPs. PDH were treated during infection with 10 μM REP 2006 (B) or REP 2086 (C). PDH were fixed on day 7 postinfection with ethanol acetic acid (EAA). DHBsAg-positive hepatocytes were visualized by confocal fluorescence microscopy as described in Materials and Methods. DHBsAg-positive PDH were detected in untreated DHBV-infected cells (A) and in infected, REP 2086-treated cells (C), while no DHBsAg-positive PDH were detected following treatment with REP 2006 (B).

Fig 4

Cytotoxic effect of treatment of PDH with REP 2006, REP 2031, and REP 2086. PDH were treated with the NAPs REP 2006, REP 2031, and REP 2086 (0.001 to 10 μM) for 7 days before addition of XTT as described in Materials and Methods. OD values were reflective of the number of viable cells present to convert the XTT salt to an orange-colored product, XTT-formazan. OD was measured at 450 nm, and plots are mean ODs ± standard deviations (SD) from 4 wells.