Nigel Bourne

Multiple Effects of Silymarin on the Hepatitis C Virus Lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarins antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA‐dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti‐HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV‐infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell‐to‐cell spread of virus. Conclusion: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarins antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell. HEPATOLOGY 2010

PSI-7851, a Pronucleotide of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine Monophosphate, Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

ABSTRACT The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine-5′-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ± 0.050 μM (mean ± standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 μM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor: Reduced RNA replication fitness and partial rescue by second-site mutations

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P′-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors

The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV isxa0infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviralxa0screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies.

Novel Imino Sugar Derivatives Demonstrate Potent Antiviral Activity against Flaviviruses

ABSTRACT Imino sugars, such as N-butyl-deoxynojirimycin and N-nonyl-deoxynojirimycin (NNDNJ), are glucose analogues that selectively inhibit cellular α-glucosidase I and II in the endoplasmic reticulum and exhibit antiviral activities against many types of enveloped viruses. Although these molecules have broad-spectrum antiviral activity, their development has been limited by a lack of efficacy and/or selectivity. We have previously reported that a DNJ derivative with a hydroxylated cyclohexyl side chain, called OSL-95II, has an antiviral efficacy similar to that of NNDNJ but significantly less toxicity. Building upon this observation, a family of imino sugar derivatives containing an oxygenated side chain and terminally restricted ring structures were synthesized and shown to have low cytotoxicity and superior antiviral activity against members of the Flaviviridae family, including bovine viral diarrhea virus, dengue virus (DENV), and West Nile virus. Of particular interest is that several of these novel imino sugar derivatives, such as PBDNJ0801, PBDNJ0803, and PBDNJ0804, potently inhibit DENV infection in vitro, with 90% effective concentration values at submicromolar concentrations and selectivity indices greater than 800. Therefore, these compounds represent the best in their class and may offer realistic candidates for the development of antiviral therapeutics against human DENV infections.

Early Production of Type I Interferon during West Nile Virus Infection: Role for Lymphoid Tissues in IRF3-Independent Interferon Production

ABSTRACT Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-α/β). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3−/−) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly α).

Construction and characterization of a second-generation pseudoinfectious West Nile virus vaccine propagated using a new cultivation system

Safer vaccines are needed to prevent flavivirus diseases. To help develop these products we have produced a pseudoinfectious West Nile virus (WNV) lacking a functional C gene which we have named RepliVAX WN. Here we demonstrate that RepliVAX WN can be safely propagated at high titer in BHK cells and vaccine-certified Vero cells engineered to stably express the C protein needed to trans-complement RepliVAX WN growth. Using these BHK cells we selected a better growing mutant RepliVAX WN population and used this to generate a second-generation RepliVAX WN (RepliVAX WN.2). RepliVAX WN.2 grown in these C-expressing cell lines safely elicit strong protective immunity against WNV disease in mice and hamsters. Taken together, these results indicate the clinical utility of RepliVAX WN.2 as a vaccine candidate against West Nile encephalitis.

Impact of Immunization with Glycoprotein D2/AS04 on Herpes Simplex Virus Type 2 Shedding into the Genital Tract in Guinea Pigs That Become Infected

In recent clinical trials, a vaccine that contained herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) and the adjuvant AS04 afforded HSV-seronegative women significant protection against HSV-2 genital disease and limited protection against infection. Similarly, in guinea pigs, immunization with the vaccine provided significant protection against genital HSV-2 disease but did not prevent mucosal infection. We explored the impact of immunization on the magnitude of latent virus infection and on the frequency and magnitude of virus reactivation as measured by both recurrent disease and viral shedding into the genital tract. Guinea pigs immunized with gD2/AS04 were shown by quantitative polymerase chain reaction (qPCR) analysis to have significantly less latent viral DNA in the ganglia than did naive control guinea pigs and to have a reduced incidence and frequency of recurrent disease. By contrast, all immunized guinea pigs shed virus into the genital tract with a frequency comparable to that seen in control guinea pigs. However, the amount of virus shed was significantly reduced, as measured by qPCR. These data suggest that immunization could affect transmission by altering viral shedding patterns.

Antiviral Profiles of Novel Iminocyclitol Compounds against Bovine Viral Diarrhea Virus, West Nile Virus, Dengue Virus and Hepatitis B Virus

The antiviral activity of iminocyclitol compounds with a deoxynojirimycin (DNJ) head group and either a straight chain alkyl or alkylcycloalkyl group attached to the nitrogen atom have been tested in vitro against multiple-enveloped viruses. Several of these analogues were superior to previously reported DNJ compounds. Iminocyclitols that inhibit the glycan-processing enzyme endoplasmic-reticular glucosidase have been shown to inhibit the morphogenesis of viruses that bud from the endoplasmic reticulum (ER) at non-cytotoxic concentrations. Bovine viral diarrhoea virus (BVDV) has been used as a surrogate system for study of the hepatitis C virus, which belong to the virus family (Flaviviridae) as West Nile virus (WNV) and dengue virus (DV). N-Nonyl-DNJ (NNDNJ) was previously reported to have micromolar antiviral activity against BVDV, but a limiting toxicity profile. N-Butylcyclohexyl-DNJ (SP169) was shown to be as potent as NNDNJ in assays against BVDV and less toxic. However, it was inactive against hepatitis B virus (HBV). The present study reports efforts to improve the performance profiles of these compounds. Introduction of an oxygen atom into the N-alkyl side chain of DNJ, either as an ether or a hydroxyl functionality, reduced toxicity but sacrificed potency. Introduction of a hydroxyl group at the tertiary carbon junction of the cycloalkyl and linear alkyl group, as in N-pentyl-(1-hydroxycyclohexyl)-DNJ (OSL-95II), led to a structure that was as well tolerated as DNJ (CC50>500 µM), but retained micromolar antiviral activity against all ER morphogenesis budding viruses tested: BVDV, WNV, DV and HBV. The implication of this modification to the development of broad-spectrum antiviral agents is discussed.

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

Background: Fibroblast growth factor 14 (FGF14) binds to and regulates the voltage-gated Na+ (Nav) channel. Results: Inhibition of glycogen synthase kinase 3 (GSK3) modifies FGF14/Nav channel interaction, with effects on Na+ currents and subcellular distribution of the FGF14·Nav channel complex. Conclusion: The FGF14·Nav channel complex is a new target of GSK3. Significance: We provide evidence for modulation of Nav channels by GSK3 through FGF14. The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na+ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na+ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.