Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry

Abstract

Background & aims: Hepatitis C virus (HCV) gains entry into susceptible cells by interacting with cell surface receptor(s). Viral entry is an attractive target for antiviral development because of the highly conserved mechanism.

Methods: HCV culture systems were used to study the effects of phosphorothioate oligonucleotides (PS-ONs), as amphipathic DNA polymers (APs), on HCV infection. The in vivo effects of APs were tested in urokinase plasminogen activator (uPA)/severe combined immunodeficient (SCID) mice engrafted with human hepatocytes.

Results: We show the sequence-independent inhibitory effects of APs on HCV infection. APs were shown to potently inhibit HCV infection at submicromolar concentrations. APs exhibited a size-dependent antiviral activity and were equally active against HCV pseudoparticles of various genotypes. Control phosphodiester oligonucleotide (PO-ON) polymer without the amphipathic structure was inactive. APs had no effect on viral replication in the HCV replicon system or binding of HCV to cells but inhibited viral internalization, indicating that the target of inhibition is at the postbinding, cell entry step. In uPA/SCID mice engrafted with human hepatocytes, APs efficiently blocked de novo HCV infection.

Conclusions: Our results demonstrate that APs are a novel class of antiviral compounds that hold promise as a drug to inhibit HCV entry.

Figure 1

Effect of PS-ON on HCV infection. (A) Huh7.5 cells were infected with HCVcc in the presence of various concentrations of 40mer PS-ON or PO-ON (degenerate sequence). Two days after infection, infected cells were detected by immunofluorescence assay using anticore antibodies (left panel). Percentage of infection was determined by dividing the number of HCV-expressing cells in treated over the untreated cells (right panel). The intracellular HCV RNA levels (B) and HCV core Ag (C) and supernatant HCV RNA (D) levels in the culture medium were determined. Hep3B cells were infected with (E) HCVpp genotype 1b or (F) VSVGpp and treated with various concentrations of PS-ON and PO-ON, and lucif-erase activities were determined 2 days later. Results are shown as percentages of infection + standard deviations (SD).

Figure 2

Sequence-independent and size- and phosphorothioation-dependent effects of PS-ON on HCV infection. A series of 40mer PS-ONs with specific sequences including poly poly G, A, T, C, TG AC, TC, and AG were tested for their inhibitory effect on HCV infection. (A) HCVcc was inoculated with Huh7.5 cells and treated with 100 nmol/L of these homo- and heteropolymeric PS-ONs. Expression of HCV core was detected by immunofluorescence assay using anticore antibodies. (B) The HCV core Ag titers and (C) HCV RNA levels in the culture medium were determined. (D) Hep3B cells were infected with HCVpp geno-type 1b and treated with these various PS-ONs at 100 nmol/L, and luciferase activities were determined 2 days later. (E) Various sizes of PS-ON (10 – 80mers) at 100 nmol/L were tested in the HCVcc and HCVpp systems. (F) Various structures of oligonucleo-tides, PS-ON analogue with phosphorothioate backbone but without the sugar or base, and PS-ON analogue with 2=-O-methyl ribose modification, were synthesized. Each 40mer oligo-nucleotide at 100 nmol/L was tested in the HCVcc and HCVpp systems. All results are shown as percentages of infection + SD.

Figure 3

Effects of PS-ON on infection of various HCV genotypes, HCV replication, and cell binding. (A) HCVpp harboring E1/E2 glycoproteins from genotypes 1a, 1b, 2a, 3a, 4a, 5a, and 6a were inoculated into Hep3B cells and simultaneously treated with 100 nmol/L of degenerate PS-ON and PO-ON (40mer). Luciferase activities were determined 2 days later. (B) Subgenomic RNA of genotype 1b Con1 or 2a JFH1 were transfected into Huh7.5 cells. Four hours after transfection, a set of transfected cells was harvested as a control for transfection efficacy, and the remaining cells were treated with 100 nmol/L of PS-ON and PO-ON. Cells were then harvested at 72 hours posttransfection and luciferase activities determined. The replication level was presented as the ratio of the luciferase activity of the sample at 72 hours over that of 4 hours. Percentages of replication were determined by dividing the replication level of treated over that of untreated samples. (C) Hep3B and Huh7.5 cells were incubated with 20 μg/mL HCV-LP and 100 nmol/L PS-ON or PO-ON at 4°C for 1 hour. The cells were washed and incubated with anti-E2 ALP98 monoclonal antibody for 30 minutes followed by FITC-labeled goat anti-mouse immunoglobulin for 30 minutes at 4°C. HCV-LP binding was analyzed by flow cytometry. The black filled peaks are negative controls without the anti-E2 antibody. The gray filled peaks are positive controls showing HCV-LP binding without any compounds. The black solid lines and gray dotted lines represent treatments with PS-ON and PO-ON, respectively. The gray solid line represents samples in the presence of HCV serum that has been shown previously to inhibit HCV-LP binding. The mean fluorescence intensity (MFI) of each sample is shown. (D) HCVcc was incubated with Huh7.5 cells in the presence of HCV serum PS-ON or PO-ON at 4°C for 1 hour. The unbound virus was washed off, and the bound HCVcc was determined by HCV RNA quantification and HCV core Ag assay. (E) HCVcc was incubated with Cy3-labeled degenerate PS-ON or PO-ON (40mer) and subjected to iodixinol density gradient analysis as described in the online Supporting Document. Control preparation generated the same way was used for comparison. The fluorescence intensity of the fraction where infectious HCV sedimented was determined and shown.

Figure 4

Effects of PS-ON on HCV viral entry. (A) Hep3B cells were incubated with HCVpp at 4°C for 1 hour to bind the virus and washed to remove the unbound virus. Cells were then incubated with fresh culture medium containing 25 nmol/L concanamycin A, 100 nmol/L PS-ON, 100 nmol/L PO-ON, or 25 μg/mL (total concentration) AP33+ALP98 monoclonal antibodies at 37°C for 16 hours. The luciferase activities were determined 24 hours later. Results are shown as percentages of infection + SD. (B) Fusion assay was performed with HCVpp or VSVGpp in the presence of PS-ON (degenerate or poly C) or the PO-ON controls. The results are expressed as mean percentages (means + SD) of inhibition of either the fusion rate at the origin of the fusion kinetics (left panel) or the maximum fusion of the curve at 500 S (right panel) relative to incubation in the absence of the compounds. The fusion curves are shown in Supplementary Figure 4. *P < .05 comparing the PS-ON and the corresponding PO-ON in the HCVpp fusion assay.

Figure 5

Effects of PS-ON on HCV infection in vivo. Human hepatocytes-transplanted uPA/SCID mice were treated intraperitoneally with 10 mg/kg of PS-ON (poly C) (n = 7) or (poly AC) (n = 5) on days −1, 0, 1, 3, 5, and 7 (indicated by dots). The corresponding control PO-ON (poly C) was also tested (n = 7). A fourth group of mice did not receive any compounds (n = 15). The mice were intravenously inoculated on day 0 with HCV patient serum containing 3.9 × 10³ copies of HCV genotype 1b (indicated by arrow). Serum samples were obtained on days 0 (prior to HCV inoculation), 7, 14, 21, 28, and 35 for HCV RNA and human albumin determination. HCV core antigen was also measured and showed the same results as the HCV RNA determination.