Crystal Structure of Human ISG15 Protein in Complex with Influenza B Virus NS1B

Abstract

ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å . A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.Type I interferon production represents one of the first lines of defense against invading viral pathogens (1, 2). Pathogens induce the expression of hundreds of interferon-stimulated genes (ISGs), 2 many of which help establish the antiviral responses in target cells (2, 3). One of the most prominent ISGs to be induced during viral infection and the ensuing type I interferon response is the ϳ15-kDa protein ISG15. It is a member of the ubiquitin-like (UBL) protein family, which is covalently attached to target proteins to modify their functions (4 -6). As ISG15 modifies proteins in a manner similar to ubiquitylation, protein conjugation by ISG15 is termed ISGylation, and proteins are sequentially catalyzed by E1-activating enzyme Ube1L, E2-conjugating enzyme UbcH8, and E3 ligases such as human HerC5 and mouse HerC6 (5-7). ISG15 consists of two UBL domains linked by a short linker. Both domains share sequence and structural similarities to ubiquitin and are required for efficient conjugation of ISG15 to target proteins (8, 9).ISG15 has recently emerged as an important cellular factor against different viral pathogens (10, 11). The antiviral activities associated with cellular and viral protein ISGylation in vitro and/or in vivo have been reported for both DNA and RNA viruses, including influenza A and B, Sindbis, herpes simplex virus type I (HSV-1), and murine ␥ herpesvirus 68 (␥HV68) (12, 13). On the other side of the host-pathogen interaction, viruses evolve differe...

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