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

Abstract

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation

“…Among the IFN-related differentially expressed genes, the upregulation of the ISG15 pathway is particularly remarkable. Earlier investigations into the role of ISG15 in HBV infection mostly suggested a proviral role although these were often overexpression studies 51–53 . Consequently, a specific antiviral role for the ISG15 pathway in the context of CAM-A treatment cannot be excluded.…”

“…Earlier investigations into the role of ISG15 in HBV infection mostly suggested a proviral role although these were often overexpression studies. [51][52][53] Consequently, a specific antiviral role for the ISG15 pathway in the context of CAM-A treatment cannot be excluded. Interestingly, Lucifora et al [54] identified upregulation of ISG15 and other factors in the ISG15 pathway (ISG15, UBE2L6, USP18, and HERC5) when inducing high HBV replication in HepaRG cells through a baculovirus-HBV system, which was associated with an antiviral state.…”

Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx

“…Previous studies have reported that NQO1 could regulate the proteasomal degradation of HBx, thereby inhibiting HBV cccDNA activity [ 17 ]. Other studies have reported that intracellular restriction genes, including TRIM21, ISG15, and TRIM31, could eliminate HBx [ 18 , 19 , 20 ]. Another inhibition strategy against HBx could include short hairpin RNAs against HBx [ 21 ].…”

ISG15 driven cellular responses to virus infection

“…impact of ISG15 conjugation or binding to viral factors to inhibit infection has been extensively reviewed elsewhere [36][37][38], which points toward its antiviral nature. Interestingly, more recent studies have also revealed a pro-viral function of ISG15 in replication of Hepatitis B [39] and C viruses [40]. These anecdotal reports indicate that co-evolution of viruses with their host may have enabled these pathogens to exploit ISGylation to their own benefit.…”