Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways

Abstract

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 ...

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

“…For the induction by Poly I:C (Sigma) or LPS (Sigma), cells were directly incubated with 5 ml of FCS-free medium containing 100 mg ml À1 of Poly I:C or 50 mg ml À1 of LPS, respectively. CAB cells were harvested at 1, 3,6,8,12,24,48, and 72 h post treatment, and total RNA was extracted with RNA-Solv Reagent (Omega Biotek). Contaminating genomic DNA in the RNA samples was thoroughly digested by DNase without RNase (Promega) before RNA was treated by chloroform and subsequently precipitated by ethanol.…”

“…Through identification of ISG15 target proteins from IFN treated cells [7,8,11,12], ISG15 and ISG15 conjugation were suggested to relate to diverse cellular pathways including RNA splicing, chromatin remodelling/polymerase II transcription, cytoskeleton organization and regulation, stress responses and translation [12]. Several recent reports have provided indirect and direct evidence for involvement of ISG15 and protein ISGylation in innate antiviral response.…”

Deficiency of a potential 3p21.3 tumor suppressor gene UBE1L (UBA7) does not accelerate lung cancer development in K-rasLA2 mice

“…Consistently, Ube1l knockout (KO) mice are deficient in ISGylation [13]. An array of essential proteins with diverse biological functions are targeted by ISGylation, underlining the biological importance of ISGylation [14,15,16,17]. Intriguingly, Ube1l KO mice appear to be fertile with no obvious phenotypes.…”

Identification and Herc5-mediated ISGylation of novel target proteins

“…The expression of ISG15 is induced by interferon stimulation and ISG15 is conjugated to various cellular proteins (ISGylation) in a manner similar to ubiquitination that is catalyzed by the sequential action of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) [4,5]. Target proteins modified with ISG15 [6][7][8], the E1 (UBE1L) and E2 (UbcH8) enzymes functioning in ISGylation [9][10][11], and a de-ISGylating enzyme (UBP43) [12] have been identified, but biological consequences of ISGylation have been studied in only a few cases [13][14][15]. Recently, Efp and Herc5 have been reported to function as E3 ligases for ISGylation [16,17], but there seems to be a difference in function between Efp and Herc5 because Herc5, but not Efp, influences the ISGylation status of whole cellular proteins [16,17].…”