The ISG15 Conjugation System Broadly Targets Newly Synthesized Proteins: Implications for the Antiviral Function of ISG15

Abstract

Summary ISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Herc5, the major E3 enzyme for ISG15, mediates the ISGylation of over 300 proteins in interferon-stimulated cells. In addressing this broad substrate selectivity of Herc5, we found that: 1) the range of substrates extends even further and includes many exogenously expressed foreign proteins, 2) ISG15 conjugation is restricted to newly synthesized pools of proteins, and 3) Herc5 is physically associated with polyribosomes. These results lead to a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner. This represents a novel mechanism for conjugation of a Ubl and further suggests that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, we demonstrate that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses.

Structural basis for the sequence-specific recognition of human ISG15 by the NS1 protein of influenza B virus

“…This mechanism is consistent with recent reports that ISG15 conjugation targets newly synthesized proteins and hence occurs on polyribosomes (22). However, in human cells transfected with a plasmid expressing the NS1B protein, and also in influenza B virus-infected human cells, the NS1B protein relocalizes essentially all the ISG15 from the cytoplasm to intranuclear compartments known as splicing or S35 speckles (16,25).…”

“…A predicted model of the ISG15-UbE1L complex (2) also locates the E1 conjugating enzyme in a region of the ISG15 C-terminal domain that is distant from the NS1B-binding sites in the N-terminal and linker domains of ISG15 identified in our crystal structure. In addition, the C-Ubl by itself also supports efficient ISG15 conjugation in human cells, as assayed by transfection experiments (22), showing that the N-Ubl has little or no role in the conjugation steps catalyzed by not only the E1 and E2 enzymes but also the E3 (Herc5) ligase. This result indicates that the Herc5 enzyme may be expected to function with the C-Ubl in an ISG15: NS1B complex, but this prediction cannot be tested at present because a functional Herc5 enzyme is not currently available.…”

“…In vitro assays have shown that the C-Ubl of human ISG15 alone supports efficient activation by the E1 Ube1L enzyme, as well as efficient transfer to the E2 UbcH8 enzyme (21). In addition, transfection experiments have recently shown that the C-Ubl of ISG15 alone supports efficient ISG15 conjugation in cells (22), demonstrating that the N-Ubl has little or no role in the conjugation steps catalyzed by the E1, E2, and E3 (Herc5) enzymes. Considering that the C-Ubl alone can function effectively with all three conjugation enzymes in cells, in order to understand how binding by NS1B inhibits ISG15 conjugation, it is important to determine if accessibility of the C-Ubl is affected by complex formation.…”

Interferon Stimulated Gene - ISG15 is a Potential Diagnostic Biomarker in Oral Squamous Cell Carcinomas

“…The ISGylation process targets more than 300 substrate proteins that includes a large number of constitutively expressed proteins regulating diverse cellular pathways such as RNA splicing, chromatin remodeling/polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation (Durfee et al, 2010). Hence a deregulation in the expression of ISG15 may be expected to promote carcinogenesis as ISGylated proteins remain stabilized for a sustained period.…”

Cellular defence or viral assist: the dilemma of HDAC6

“…Additionally, HDAC6 can restrict viral spread by interacting with interferon-stimulated gene 15 (ISG15) and ISG15-linked proteins, targeting them for selective autophagic degradation [112]. Previous work has demonstrated that ISG15 knockout mice are broadly susceptible to RNA and DNA viruses [113]. More detailed study has shown that ISG15 could broadly conjugate IFN-induced ISGs and viral proteins, mediating their degradation during the process of virion egress and release.…”