Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Palo Alto, CA 94305-5187, USA
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Abstract |
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Keywords: farnesyltransferase inhibitors, hepatitis delta virus, antiviral therapy
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Prenylation as a post-translational modification of proteins |
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Prenylation in HDV |
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HDV has a single-stranded circular RNA genome. The latter encodes two proteins, the small and large hepatitis delta antigens, which are identical except for the presence of an additional 19 amino acids at the C-terminus of the larger isoform.6 The complete virus particle is composed of a complex of genome and both delta antigen isoforms, which is encapsulated by a lipid envelope embedded with HBV surface antigen (HBsAg) envelope proteins.7 As the latter are not encoded by HDV, but rather by a co-infecting HBV, this provides a molecular explanation for why HDV infection occurs only in the presence of a co-existing HBV infection. Although they share most of their amino acid sequence in common, the two delta antigen isoforms have remarkably different functions. For example, while the small delta antigen is essential for genome replication, the large delta antigen (LHDAg) can trans-dominantly inhibit this process8,9 and can transactivate a variety of genes.10 With respect to HDV particle assembly, only the LHDAg can mediate assembly and release of HBsAg-enveloped particles.11 The critical molecular determinants for HDV particle assembly were first determined using the simplest model of HDV assembly, virus-like particles (VLPs). The latter consist of LHDAg and small surface antigen (the smallest of the three HBV envelope proteins), which together are the minimal elements required for particle assembly.
Within the 19 C-terminal amino acids unique to the larger delta antigen isoform, a CXXX box motif has been identified.12 This motif, comprising the last four amino acids of LHDAg, was noted to be absolutely conserved across HDV isolates, suggesting that it reflected an important function in vivo. It was hypothesized that this conserved CXXX box was a substrate for prenylation and that such a lipid modification could help mediate interaction with the membrane-associated HBsAg required for HDV morphogenesis. Labelling studies with [3H]mevalonatethe metabolic precursor of prenyl lipidsshowed that LHDAg was indeed subject to prenylation both in in vitro translation reactions12 and in intact cells.12,13 Site-directed mutagenesis demonstrated that genetic disruption of the CXXX boxsuch as by substitution of the CXXX box cysteine with serineabolished both prenylation of LHDAg and its ability to interact with,13 and form secreted particles with, HBsAg.12 This was the first demonstration that a viral protein could be modified by prenylation, and highlighted a novel mechanism of virion assembly. The specific type of prenyl lipid added to LHDAg was shown to be farnesyl.14
Although attempting to genetically disrupt the LHDAg CXXX box would not be practical in natural infections, if a similar end resultnamely prevention of prenylationcould be achieved pharmacologically (Figure 1b), these results could indeed be translated into a practical clinical strategy. As detailed below, this approach has been progressively evaluated, first with VLPs, then complete infectious virions, and most recently in an in vivo mouse model of HDV.
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Prenylation inhibition-based inhibition of HDV assembly |
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Subsequently, the effect of another farnesyltransferase inhibitor, FTI-277, was evaluated in a system that produces complete, infectious HDV virions containing an intact genome.17 This system was based on co-transfecting Huh7 cells with plasmids encoding the complete HDV and HBV genomes.18 Mid-nanomolar concentrations of FTI-277 dramatically inhibited virion production, and low micromolar concentrations of the compound decreased virion production to below the level of detection.17 Again, no effect on general protein synthesis and secretion or cell metabolism was exhibited. The significant difference between the molecular structures of BZA-5B and FTI-277 suggested that HDV assembly was indeed inhibited by their common FTI activity rather than some other feature of the inhibiting drugs. Thus in spite of the added complexity and assembly determinants of infectious HDV virions compared with VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions that are associated with particularly severe clinical disease19 was as sensitive to prenylation inhibition as was that of HDV genotype I virions.17
Most recently, the antiviral efficacy of FTIs was evaluated in vivo in a new mouse model of HDV. This model was established by hydrodynamically transfecting HBV-transgenic FVB mice with HDV-encoding plasmids, leading to robust intrahepatic viral replication and viraemia.20 Cohorts of mice in which HDV viraemia had been established were then treated with single daily doses of the prenylation inhibitors FTI-277 or FTI-2153, or vehicle controls. Both agents were highly effective at clearing HDV viraemia. As expected, HDV inhibition exhibited duration of treatment dependence. In addition, similar alanine aminotransferase levels among treatment groups argued against a non-specific hepatotoxic effect of the FTIs as a potential cause for the clearance of viraemia.20 This is a dramatic and clear first in vivo confirmation of the potential of this novel class of antiviral agents. These results have obvious clinical relevance and importance for human HDV infections. Since, in the mouse model, newly produced virions cannot infect new hepatocytes, the effect of FTIs on viral-related liver injury cannot be evaluated fully. In human HDV infections, however, it is expected that inhibition of the critical steps of assembly and release in the virus life cycle would have a major impact on the course of HDV infection and its associated liver disease.
At least for chronic HDV infection, because of the dependence on HBV for providing a source of HBsAg, theoretically, effective eradication of HBV in an individual would be expected to eventually lead to clearance of HDV as well. Unfortunately, the currently widely used oral anti-HBV agent lamivudine, while effectively decreasing HBV DNA levels, leaves HBsAg largely unaffected;21 as such it would be predicted to have little effect on HDV. Indeed, when used alone or in combination with high-dose interferon, lamivudine does not improve disease activity or lower HDV-RNA levels in patients with chronic delta hepatitis.22,23
Because of the lack of an effective anti-HDV therapy and the promising preclinical data in mice, FTIs are currently being considered for trials in human patients. For such purposes, well-tolerated, orally available FTIs would be preferred. Fortunately, the requisite candidate drugs have already been developed and used in humans, albeit for a different purpose. The requirement for prenylation in transformation by oncogenic Ras has made the enzymes responsible for this modification important targets for antitumour drug design. A variety of prenylation inhibitors have been synthesized and been evaluated for clinical use in several Phase I/II and III trials.24 These studies provide important information when considering this class of drugs as antiviral agents. First, most studies have used oral regimens, emphasizing a convenient route of administration. Secondly, the use of FTIs was found to generally be quite safe, with collectively the main reported side effects being reversible, dose-dependent myelosuppression, fatigue, reversible neurocortical toxicity, prolongation of QT and mild gastrointestinal toxicity. Moreover, side effects differed depending on the particular FTI used, suggesting the side effects might be more compound-related rather than a direct effect of inhibiting farnesylation.
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Attractive features of FTIs and application to other viruses |
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The described strategy could affect host cell prenylation as well, theoretically causing intolerable side effects. Surprisingly, this does not appear to be the case, as FTIs are remarkably well tolerated by host cells in vitro,25 and more importantly, treated cancer patients in vivo.26 Perhaps this reflects the fact that most prenylated proteins in cells are modified by geranylgeranyl rather than farnesyl27 or that the existence of a family of prenyltransferase enzymes offers the possibility of cross prenylation when a particular prenyltransferase is targeted by drug treatment.
Prenylation inhibition-based antiviral therapy has implications for other viruses besides HDV, which are found to have similarly prenylated proteins. Analysis of sequence databanks reveals that a CXXX box motif is present in proteins of numerous other medically important viruses, as well as in agents with a potential for bioterrorism. For example, UL32, a gene product of herpes simplex virus (HSV), thought to be involved in virus particle formation, contains such a CXXX box.28 Interestingly, Farassati et al.29 have treated a Ras-transformed cell line infected with HSV-1 with FTI-1 in order to evaluate whether Ras is involved in HSV-1 infection. A significant decrease in HSV-1 titres in treated cells as well as decreased viral protein synthesis were demonstrated, leading to their conclusion that HSV-1 exploits the host cell Ras signalling pathway for infection.29 Although not specifically evaluated, treatment with FTI-1 may also have had more of a direct antiviral effect of inhibiting viral protein prenylation, contributing to the observed viral inhibition. This might suggest that in HSV-1, and possibly in other viruses, prenylation inhibitors could have a dual mechanism: inhibiting prenylation of a viral protein as well as the cellular pathways exploited by the virus.
Prenylation is also predicted to be involved in viral processes beyond assembly. For example, in hepatitis A virus and the animal enterovirus causing foot and mouth disease,30 it is the polymerase protein that bears a CXXX box. Because these viruses replicate their RNA in intimate association with host cell membranes, prenylation may help mediate membrane-associated RNA replication. Not all CXXX boxes undergo prenylation.1 Thus, formally, the identification of a CXXX box in the protein of a contemplated viral target should be complemented by direct demonstration that the protein is indeed subject to prenylation. Finally, motifs that mediate other types of prenylation in host cell proteins such as terminal CC and CXC are expected to be operational in viral counterparts as well.
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Other targets for antiviral therapy in the prenylation pathway |
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To summarize, prenylation inhibitors target a host cell function, are available in oral form and are surprisingly well tolerated in human trials to date. Although first shown to be effective against HDV, a large number of other viruses appear to contain proteins that are subject to prenylation. Thus, prenylation inhibitors represent an attractive new class of antiviral agents with potential for broad-spectrum activity.
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Acknowledgements |
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Footnotes |
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References |
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