Modulation of alpha interferon anti-hepatitis C virus activity by ISG15.

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-alpha) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-alpha, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-alpha-specific effect, which was not observed with IFN-gamma, correlated with an increase in expression of the IFN-alpha-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-alpha-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-alpha after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-alpha-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-alpha treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-alpha pathway and its antiviral effect.

Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients.

OBJECTIVES: To characterize the effect of hepatitis C virus (HCV) polymerase intrinsic genetic heterogeneity on the inhibitory activity of nucleoside and non-nucleoside HCV polymerase inhibitors. METHODS: The sensitivity of genotype (GT) 1 HCV NS5B clinical isolates from treatment-naive patients to nucleoside and non-nucleoside polymerase inhibitors was assessed. The genetic diversity at the population level, as well as that of their quasispecies, was correlated with the observed reduced sensitivity to inhibitors. RESULTS: R1479 and NM107 (nucleoside analogues that have entered Phase 2 clinical trials as prodrugs R1626 and NM283, respectively) were similarly active across the tested clinical isolates. Resistance mutations to nucleoside analogues were not observed in any of the isolates. However, the activity of the non-nucleoside thumb II inhibitor NNI-1, palm I inhibitors NNI-2 and NNI-3, and palm II inhibitor HCV-796 was reduced across different isolates. This reduction in inhibitory activity for non-nucleoside inhibitors (NNIs) was, in most cases, correlated with the existence of known NNI resistance mutations in the NS5B polymerase population of the clinical isolates, as detected by population sequencing. Resistance mutations to NNIs were also observed at a low frequency within the clinical isolates' viral quasispecies that allowed for their rapid selection upon drug selective pressure. CONCLUSIONS: The higher frequency of known NNI resistance mutations or polymorphisms known to affect their antiviral potency when compared with the lack of detection of resistance mutations to the nucleoside analogues suggests a potential for primary reduced responsiveness as well as faster development of clinically significant resistance.

CCR5 small-molecule antagonists and monoclonal antibodies exert potent synergistic antiviral effects by cobinding to the receptor.

A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.