Current therapy for hepatitis C is based on peginterferon and ribavirin. However, the therapy is only effective in approximately half of the patients, and there is little option to those who fail current therapy. Recent advances in the development of small molecule inhibitors targeting the viral-encoded enzymes showed promise,27
but viral resistance to these drugs is a major clinical issue because HCV is highly variable with rapid viral proliferation and low-fidelity replication. Phosphorothiate modification of oligonucleotides was initially designed to reduce enzymatic degradation. This modification also increases the hydrophobicity of the phosphodiester backbone and thus imparts an amphipathic character to the oligonucleotide polymer.28
Recent studies showed that the amphipathic PS-ONs have a sequence-independent antiviral activity against HIV-1 and other viruses,14,29
suggesting that these compounds may exhibit a broad-spectrum antiviral activity.
Our data showed that PS-ON blocked HCV infection in the HCVcc and HCVpp systems in a similarly dose-dependent manner, with 50% inhibitory concentration in the nanomolar range. PS-ON had no effect on infection of VSVGpp (an enveloped RNA virus with mechanism of viral entry distinct from type I and II fusion) or adeno-virus (a nonenveloped DNA virus). The amphipathic nature of PS-ON is crucial for its anti-HCV property because PS-ON analogs lacking the amphipathicity are inactive. Polynucleotides are polyanions, a class of compounds that have been shown to interfere with a variety of viral infections.30,31
However, our data showed clearly that the polyanionic nature is not relevant to the PS-ON inhibitory activity because the control PO-ON is not active in HCV inhibition. Furthermore, the inhibitory effect of PS-ON could not be explained by the increased stability of the phosphorothioation because the control PO-ON has the 2′-O-methyl modification that also stabilizes the oligonucleotides.14,23
The inhibitory activity of APs is sequence independent but length dependent. The degenerate APs were equally effective as the homo- and heteropolymeric sequences, with the exception of poly A and G, which can form unique polypurine quartet structures in solution.32
The minimal length of PS-ON required for potent inhibitory activities is 40mer, which appears to be the same for all active PS-ON compounds. This length-specific requirement may indicate a critical structural feature of the HCV entry process that is susceptible to these compounds. Although the degenerate PS-ON may contain CpG motif, the other hetero- and homopolymer PS-ONs tested herein are devoid of CpG motifs. The comparable anti-viral activity of these compounds to the degenerate PS-ON demonstrates that the antiviral activity is not mediated by the potential CpG-mediated induction of interferon. Furthermore, Huh7.5 cells express very little or none of the cell surface toll-like receptors involved in recognition of nucleic acid-based motifs,33
and we did not observe any production of endogenous type I inter-ferons in cells exposed to either PS-ON or PO-ON.
The inhibitory activity of PS-ON appears to target the postbinding and prereplication stage and possibly at the fusion step of HCV infection. The fusion process appears to be structurally conserved among many enveloped viruses and can be classified into types I and II.34
The type I membrane fusion is exemplified by the influenza and HIV-1 via hemagglutinin and gp41, respectively. The type II fusion includes the alpha-viruses and flaviviruses.34–36
It has been proposed that HCV uses a type II fusion process because of its similarity to flaviviruses.37
Our recent study suggests that HCV and flaviviruses are indeed structurally similar.38
It is conceivable that the fusion process of HCV may be susceptible to inhibition by the amphipathic structure of PS-ON, but further confirmation is necessary. HCV entry has been shown to occur via receptor-mediated endocytosis and is sensitive to lysosomotropic agents and inhibitors of vacuolar ATPases.39
The finding that PS-ON acts at the postbinding step like concanamycin A and bafilomycin A1, which are potent inhibitors of the vacuolar ATPases, supports this hypothesis. Furthermore, all HCV genotypes appeared to be susceptible to the APs equally, suggesting that the process involved is highly conserved.
HCV entry involves multiple cellular factors, such as CD81, SR-B1, Claudin-1, heparin sulfate, DC-SIGN, and L-SIGN, and possibly LDL receptor.5–10,31
CD81 and Claudin-1 have been postulated to act on the postbinding step.6,40
SR-B1 is likely involved in an early viral entry step to the cells. Its interaction with apolipoproteins and cholesterol transfer property appear to be important for viral entry,41
possibly at the level of membrane fusion.42
The overall mechanism of HCV entry is complex and involves multiple factors and steps. The APs likely interact with 1 of these essential steps to abort HCV entry. The unique inhibitory effect of the APs on HCV infection makes it a valuable reagent to study the molecular pathway of HCV entry. The APs can also be developed as a molecular probe to image and dissect biochemically this complex process.
Our study demonstrates that APs are potent inhibitors of HCV infection. APs are equally effective against all HCV genotypes and can inhibit de novo HCV infection in the human hepatocyte-transplanted uPA/SCID mouse model. This approach has the advantage of a novel and highly conserved target mechanism that is distinct from the small molecule inhibitors being developed clinically as well as the well-established pharmacology of antisense-based nucleic acid molecules in clinical trials. The effectiveness of this class of compounds in blocking de novo HCV infection supports its value in liver transplantation to prevent reinfection, which occurs invariably and presents a major problem for the management of these patients.43
So far, prophylactic reagents based on neutralizing antibodies have been disappointing in clinical trials of liver transplantation.44
Our studies illustrate the promise of this class of compounds as a potent antiviral against HCV and support its further development in the therapy of hepatitis C.