Our interest in ISG15 originated in the course of experiments to elucidate the function of the NS1B protein of influenza B virus. We found that the NS1B protein binds ISG15 and inhibits its conjugation (6), indicating that ISG15 conjugation is likely to be an important part of the IFN-␣͞-induced antiviral response. However, it was not evident how ISG15 conjugation might serve such a role. To address this issue and to elucidate the function of ISG15 conjugation, we first identified the E1 and E2 enzymes in the ISG15 conjugation pathway as Ube1L and UbcH8, respectively, both of which are induced by IFN-␣͞ (6, 7). These findings enabled us to develop a system for a proteomics-based identification of ISG15 target proteins, which is described in the present study.We used this system to identify a large number (158) of ISG15 modified proteins in IFN--treated human (HeLa) cells. The identity of these ISG15 target proteins provides insights into the function of ISG15 modification. Several of the targets are IFN-␣͞ -induced antiviral proteins, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed human proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling͞ polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions and may have a role in regulating transcription and pre-mRNA splicing during the IFN-␣͞ response. Thus, by targeting this wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-␣͞.
Materials and MethodsPlasmids. Plasmids containing the following PCR-generated reading frames were inserted into pcDNA3 vectors: Ube1L, UbcH8, His 6 -HA-ISG15, and His 6 -3xFLAG-ISG15. All of the cDNAs used for verifying ISG15 target proteins, except maspin, were generated by PCR by using a Human Leukocyte Matchmaker cDNA library (Clontech). The template for amplifying maspin was pEF-Maspin, provided by Zhang Min (Baylor School of Medicine, Houston). For the expression of V5-tagged target proteins, two modified pcDNA3 vectors containing the V5 epitope were constructed. The original BamHI site of pcDNA3 was eliminated and replaced by the V5 sequence followed by either a BamHI site (pcDNA3-V5-Bam) or a NotI site (pcDNA3-V5-Not). The PCR-generated reading frames for maspin, PTB-1, and thioredoxin reductase-1 (TrxR1) were cloned into pcDNA3-V5-Bam as BglII-BglII, BglII-R1, and BamH-R1 fragments, respectively. The PCR-generated reading frames for Hsp60 and moesin were cloned into pcDNA3-V5-Not as Not-XbaI and Not-EcoRI fragments, respectively. For the expression of 3xFLAG-RIG-I, its PCR-generated reading frame was inserted into the pCMV10 vector (Sigma).Purification of ISG15 Conjugates. HeLa cells in each of five 150-mm culture dishes (total of 10 8 cells) were transfected by using Fugene 6 ...