Research Article: Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15

Date Published: July 4, 2008

Publisher: Public Library of Science

Author(s): Susana Guerra, Ana Cáceres, Klaus-Peter Knobeloch, Ivan Horak, Mariano Esteban, Mark L. Buller.


The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.

Partial Text

Type I interferons (IFN-α and -β) serve a critical role in antiviral innate immunity and in modulating the adaptive immune response to infection and tumor development [1]. In response to infection or Toll-like receptor agonists, IFN is produced and consequently leads to the up-regulation of hundreds of IFN-stimulated genes (ISG) [2],[3]. One of the most highly induced genes is ISG15 that encodes a small UBL protein of 17 kDa that forms covalent conjugates with cellular proteins [4]. ISG15 is composed of two domains, each of which carries high sequence and structural similarity to UB (33 and 32% for the N- and C-terminal domains, respectively) [5],[6].

Pro-inflammatory and IFN-stimulated genes (ISGs) represent essential components of the innate immune response to viral infection (40). Upon viral entry into cells, ISG induction occurs in two waves: acute, IFN-independent induction of a subset of ISGs and delayed, IFN-dependent induction via the production of IFN-α/β during the initial phase. In many viral infections, IFN-independent ISG induction is mediated by the IRF-3 phosphorylation, homodimerization, and nuclear translocation. Activated IRF3, in turn, induces the expression of type I IFN genes, whose products trigger strong induction of a subsets of ISGs, including IFN-β which after its release and ligand-binding to its receptor then initiates IFN-dependent ISG induction via the IFN receptor and JAK/STAT signaling pathways. IFN-inducible enzymes, like the 2.5 OAS/RNAse L system, PKR, and M×, are the best characterized proteins that mediate antiviral action of IFN. Another protein, ISG15, was first identified as an IFN-stimulated gene whose expression is induced strongly by IFN-α/β treatment and can be detected at low constitutive levels in cells [45]. ISG15 modifies several important molecules and affects type I IFN signal transduction; ISG15 expression is markedly increased following viral infection (14, 30, 49), and many viruses encode inhibitors of the IFN-transduction pathway or specific inhibitors of ISG to avoid deleterious effects triggered by these cytokines.