ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. We describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes — granulocytes in particular — reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of Nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.

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.

Previous studies have indicated that 6–30% of all newly synthesized proteins are rapidly degraded by the ubiquitin-proteasome system, however the relationship of ubiquitination to translation for these proteins has been unclear. We report that co-translational ubiquitination (CTU) is a robust process, with ~12–15% of nascent polypeptides being ubiquitinated in human cells. CTU products contained primarily K48-linked polyubiquitin chains, consistent with a proteasomal targeting function. While nascent chains have been shown previously to be ubiquitinated within stalled complexes (CTUS), the majority of nascent chain ubiquitination occurred within active translation complexes (CTUA). CTUA was increased in response to agents that induce protein misfolding, while CTUS was increased in response to agents that lead to translational errors or stalling. These results indicate that ubiquitination of nascent polypeptides occurs in two contexts, and define CTUA as a component of a quality control system that marks proteins for destruction while they are being synthesized.

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-␣/␤-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1 Ub (Ube1). K m determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K m difference), similar to the preference of E1 Ub for UbcH7 over UbcH8 (a 36-fold K m difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the ␣1-helix and the ␤1-␤2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1 Ub resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8. Ubiquitin (Ub)3 and ubiquitin-like proteins (Ubls) are covalently conjugated to proteins through amide bonds formed between their terminal carboxyl groups and, in most cases, ⑀-amino groups of lysine residues of target proteins. Two groups of enzymes, the E1 and E2 enzymes, are essential for all known Ub/Ubl conjugation pathways. These enzymes function cooperatively in reactions that involve enzyme-bound thioester intermediates (1). E1 enzymes catalyze Ub/Ubl activation by first forming an ATP-dependent Ub/Ubl-adenylate, followed by an enzyme-bound Ub/Ubl-thioester at the active site cysteine of the E1. The activated E1 then transfers the Ub/Ubl to the active site cysteine of specific E2 enzymes in a transthiolation reaction, preserving the Ub-thioester linkage.

ISG15 is a ubiquitin-like modifier that is expressed in response to type 1 interferon signaling (IFN-α/β) and plays a role in antiviral responses. The core E1, E2, and E3 enzymes for ISG15 are Ube1L, UbcH8, and Herc5, respectively, and these are all also induced at the transcriptional level by IFN-α/β. We recently showed that Herc5 associates with polysomes and modifies target proteins in a cotranslational manner. Here, we describe the expression of the core conjugating enzymes in human cells, the detection of ISG15 conjugates, and the methods for fractionation of Herc5 with polysomes.