Inhibition of Hepatitis C Virus Replication by IFN-Mediated ISGylation of HCV-NS5A

Abstract

ISG15 is a ubiquitin-like molecule whose expression is induced by type I IFN (IFN-α/β) or in response to virus or bacterial infection. ISG15 or conjugation of ISG15 to target proteins was reported to play critical roles in the regulation of antiviral responses. IFN restricts replication of hepatitis C virus (HCV). However, molecular mechanism of IFN-α/β that inhibits HCV replication is not clear yet. In the current study, we demonstrated that replication of HCV was inhibited by overexpression of ISG15 and ISG15-conjugation enzymes in the HCV subgenomic replicon cells. Among various nonstructural proteins of HCV, NS5A was identified as the substrate for ISGylation. Furthermore, protein stability of NS5A was decreased by overexpression of ISG15 or ISG15-conjugating enzymes. The inhibitory effect of ISG15 or ISGylation on NS5A was efficiently blocked by substitution of lysine at 379 residue to arginine within the C-terminal region, suggesting that ISGylation directly controls protein stability of NS5A. Finally, the inhibitory effect of IFN-α/β on HCV replication was further enhanced by ISGylation, suggesting ISG15 as a therapeutic tool for combined therapy with IFN against HCV.

The ISG15-specific protease USP18 regulates stability of PTEN

“…Since USP18 removes ISG15 from its target proteins, it was hypothesized that gain of ISG15 expression had a similar effect on PTEN as did USP18 loss. This possibility was supported by previous work indicating that overexpression of ISG15 decreased expression of its target proteins [29]. Indeed, engineered overexpression of ISG15 decreased PTEN levels in both murine and human lung cancer cell lines by at least ~40-50% as compared to vector control transfected cells (Figure 2C).…”

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

“…Moreover, it has been described that ISG15 expression blocks the virus-budding process by different mechanisms such the blockage of Endosomal Sorting Complexes Required for Transport (ESCRT) machinery for HIV [15], or in the case of Ebola and other enveloped viruses infections, inhibiting the Nedd4 E3 ubiquitin ligase [23]. Furthermore, several viral proteins have been shown to be conjugated to ISG15, such as the NS1 protein from influenza virus, NS5A from Hepatitis C virus [24] and gag from HIV virus [25]. It has been proposed that conjugation to viral proteins inhibits specific viral functions or virions assembly, causing a block in viral infection progression [26], [27].…”

Emergent properties of the interferon-signalling network may underlie the success of hepatitis C treatment

“…In the presence of IFN, ISGs are transcribed to produce ISG mRNA. The translation of ISG mRNA yields ISG proteins, which control HCV by inhibiting different steps of the HCV life cycle and/or lowering the stability of HCV RNA [23][24][25][26][27] . Of the ninety ISGs expressed in response to IFN (type I) stimulation, seven were recently found to exhibit significant activity against HCV 24 .…”

“…At the same time, key ISGs involved in the control of HCV have been identified and in some cases their mode of action elucidated [23][24][25][26][27] . These specific interactions between HCV and components of the IFN network have unravelled the many fronts of the battle between HCV and our innate immune system.…”