Of the several hundred proteins induced by interferon (IFN) a/b, the ubiquitin-like ISG15 protein is one of the most predominant. We demonstrate the novel way in which the function of the ISG15 protein is inhibited by in¯uenza B virus, which strongly induces the ISG15 protein: a speci®c region of the in¯uenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of ISG15 to its target proteins both in vitro and in infected cells. We identify UBE1L as the E1 enzyme that catalyzes the ®rst activation step in the conjugation of ISG15, and show that the NS1B protein inhibits this activation step in vitro. In¯uenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection. We discuss the likely basis for these different strategies.

SummaryCD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating α galactosylceramide (αGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for αGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing αGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

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Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-␣͞␤ stimulation. Although the E1 enzyme for ISG15 (Ube1L͞E1 ISG15 ) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-␣͞␤ response. IFN-␣͞␤ play an essential role in innate immunity and are induced during many types of viral infections (1). Many genes are transcriptionally induced by IFN-␣͞␤, including ISG15 (IFNstimulated gene, 15 kDa) (2, 3). The ISG15 protein is a 15-kDa ubiquitin (Ub)-like protein (Ubl), consisting of two Ub-related domains, Ϸ30% (N-terminal domain) and 36% (C-terminal domain) identical to Ub. ISG15 becomes conjugated to a diverse set of cellular proteins after IFN-␣͞␤ stimulation (4). Although the biochemical consequences of ISG15 conjugation and the fate of the conjugated proteins are not known, it does not appear that ISG15 targets proteins for proteasomal degradation (5, 6).Conjugation of Ub to target proteins requires the cooperative activities of at least three classes of enzymes (7). The ATPdependent E1 enzyme activates Ub by C-terminal adenylation, followed by formation of a high-energy thioester bond between the terminal carboxylate of Ub and the active-site cysteine of E1. Ub is then transferred to the active-site cysteine of one of a number of related E2 enzymes. E3 enzymes then promote transfer of Ub from the E2 to the substrate, resulting in a stable amide bond between -amino groups of lysine side chains and Ub. E3 enzymes are the primary determinants of substrate specificity and can be divided into two classes based on mechanism. HECT E3s accept Ub from the E2 enzyme, again in the form of a thioester adduct, and transfer Ub from their active-site cysteine to the bound substrate (8). RING E3s consist of several subclasses and are either single or multisubunit enzymes that serve as docking proteins for both protein substrates and activated E2 enzymes, with transfe...