ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

Abstract

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called 'ISGylation'. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A-ISGylation in HCV replication is pro-viral or anti-viral. Here we investigated the role of NS5A-ISGylation in HCV replication by using HCV RNA replicons that have a mutation at each lysine (Lys) residue of NS5A protein. Immunoblot analyses revealed that five Lys residues (K44, K68, K166, K215, and K308) of 14 Lys residues within NS5A (1b, Con1) have the potential to accept ISGylation. We tested the NS5A-ISGylation among different HCV genotypes and observed that the NS5A of all of the HCV genotypes accept ISGylation at the multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that the residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A-ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence suggesting that NS5A-ISGylation derived from all of HCV genotypes enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A-ISGylation functions as a pro-viral factor and promotes HCV replication via the recruitment of CypA. IMPORTANCE Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type-I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible, ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here we demonstrate that HCV NS5A protein accepts ISG15-conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A-ISGylation. We obtained evidence suggesting that NS5A-ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

“…However, immunoblot analyses indicated ISGylated-HBx protein showed more than 130 kDa, suggesting that poly-ISGylation or ISG15-ubiquitin mixed chains might be involved in HBx ISGylation. We reported similar findings in HCV NS5A ISGylation [26].…”

“…ISG15/ISGylation has been reported to play an antiviral role, but in the case of several infections -including infections with HBV and hepatitis C virus (HCV) -ISG15/ISGylation has been reported to have a pro-viral function [20,25,26]. In the case of HBV infection, Li and colleagues [25] reported that the overexpression of ISG15 and subsequent ISGylation could promote HBV production in HBV-persistent replicating cells, although the putative ISGylated viral protein(s) remain to be determined.…”

HERC5 and the ISGylation Pathway: Critical Modulators of the Antiviral Immune Response

“…Previous domain-mapping experiments have determined that the HERC5 N-terminal region contains five regulator of chromatin condensation 1 (RCC1) motifs which collectively form an RCC1-like domain (RLD)-a substructure of HERC5 that plays a direct role in the recognition and coordination of viral substrates [84]. For example, studies have shown that HERC5 uses its RLD domain to bind the influenza A virus (IAV) non-structural protein 1 (NS1) [47], the hepatitis C virus (HCV) non-structural protein 5A (NS5A) [85] and multiple human immunodeficiency virus (HIV) gag particle precursor proteins [86]. Given the expansive amount of new research being conducted on HERC5 ISGylation, it is likely that additional HERC5 pathogenic substrates will be discovered, and the immunological consequences of these interactions will need to be resolved.…”

“…HERC5 has recently been shown to carry out viral protein ISGylation to (i) stall IAV replication by blocking NS1 protein homodimerization [47], (ii) promote HCV proliferation via improved cyclophilin A recruitment by NS5A proteins [85] and, alternatively, (iii) inhibit an early stage of HIV assembly by attenuating gag-particle production [86,109]. In the upcoming sections we discuss some of the recent discoveries involving the HERC5-dependent ISGylation of viral protein targets.…”

“…Taken together, these findings suggest that NS5A is the primary HCV target substrate of HERC5-dependent ISGylation, and that K379 is the sole NS5A residue that HERC5 targets for ISGylation. More recently, Abe et al demonstrated that the HCV NS5A protein is prone to ISGylation at five Lys residues (K44, K68, K166, K215 and K308) [85]. In fact, one of the ISG15 attachment points on NS5A, K308, is located within the NS5A-binding region for cyclophilin A (CypA), a virulence factor that is required for efficient HCV cellular propagation.…”