The goal of this study was to elucidate the molecular mechanism by which type I IFN inhibits assembly and release of HIV-1 virions. Our study revealed that ISG15 was one of the first recognized ISGs (4). ISG15 is covalently conjugated to targeted proteins through a series of steps similar to ubiquitin conjugation. The ISG15 activating enzyme is ubiquitin E1 like protein (UBE1L), and the major E2 enzyme for ubiquitin conjugation, UbcH8, also recognizes ISG15 (5). An ISG15-specific ligase E3 has not yet been identified, but the IFN-induced Ub E3-like enzymes Rsp5 and CEB1͞Hc5 may function in ISG15 conjugation (6, 7). Like Ub (8), ISG15 is removed from conjugated proteins by an ISG15-specific protease, UBP43 (9). Interestingly, type I IFN stimulates expression not only of ISG15 but also UBEL1, UbcH8, and UBP43 (3, 10) and markedly increases ISG15 conjugation. ISG15 targets a large number of cellular proteins (7); however, modification by ISG15 does not typically cause substrate degradation (11).Ub is a central cellular regulator (12), and Ub-mediated proteolysis plays a regulatory role in the immune system (13). In general, polyubiquitination targets proteins toward 26S protease-associated degradation, whereas monoubiquitination is a signal for internalization and vesicle sorting. However, whereas polyubiquitination on lysine 43 targets proteins for degradation, lysine 63 polyubiquitination is a signal for kinase activation. Many viruses modulate the Ub-proteasome pathway to alter cellular signaling and the antiviral response (14, 15). HIV-1 uses ubiquitination at two steps of its replication cycle. First, HIV-1-encoded protein Vif targets cellular cytidine deaminase APOBEC3G for Ub mediated degradation, thus preventing APOBEC3G incorporation into viral particles and deamination of the viral genome during reverse transcription (16-18). Vif itself is monoubiquitinated and this may help its recruitment to the site of viral assembly (19). Second, ubiquitination of Gag by Ub ligase Nedd4.1 is critical for assembly and release of virions from infected cells (20). HIV-1 assembly is driven by the Gag poly protein (21), and deletion of the PTAP-L domain in p6 of Gag, or inhibition of the Gag interaction with Tsg101, a protein of the endosomal sorting complex (ESCRT-I) (22), results in accumulation of HIV-1 virions at the plasma membranes.An important role for type I IFN in the innate response to HIV-1 infection is suggested by the observation that deletion of high IFN producing dendritic pDC2 cells results in rapid progression of HIV-1 infection in vivo (23). In vitro, type I IFN inhibits HIV-1 infection both at early steps of replication (24,25) and at the late steps of virus assembly and release (26-28). HIV-1 virions assembled in IFN-treated T cells show a low infectivity, accumulate at the plasma membrane, and have altered morphogenesis (29)(30)(31). Similar defects were seen in IFN-treated cells infected with murine leukemia virus (32). We have shown that IFN-␣-and IFN-␤-mediated inhibition correlates with the induct...

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