The Interferon-Induced Gene ISG15 Blocks Retrovirus Release from Cells Late in the Budding Process

Abstract

The release of retroviruses from cells requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including the ESCRT-III proteins and the Vps4 ATPase. In response to infection, cells have evolved an interferon-induced mechanism to block virus replication through expression of the interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin, which interferes with ubiquitin pathways in cells. Previously, it has been reported that ISG15 expression inhibited the E3 ubiquitin ligase, Nedd4, and prevented association of the ESCRT-I protein Tsg101 with human immunodeficiency virus type 1 (HIV-1) Gag. The budding of avian sarcoma leukosis virus and HIV-1 Gag virus-like particles containing L-domain mutations can be rescued by fusion to ESCRT proteins, which cause entry into the budding pathway beyond these early steps. The release of these fusions from cells was susceptible to inhibition by ISG15, indicating that there was a block late in the budding process. We now demonstrate that the Vps4 protein does not associate with the avian sarcoma leukosis virus or the HIV-1 budding complexes when ISG15 is expressed. This is caused by a loss in interaction between Vps4 with its coactivator protein LIP5 needed to promote the formation of the ESCRT-III-Vps4 double-hexamer complex required for membrane scission and virus release. The inability of LIP5 to interact with Vps4 is the probable result of ISG15 conjugation to the ESCRT-III protein, CHMP5, which regulates the availability of LIP5. Thus, there appear to be multiple levels of ISG15-induced inhibition acting at different stages of the virus release process.

The IFITM Proteins Inhibit HIV-1 Infection

“…BST-2 blocks the release of HIV-1 particles from the cell surface (44,64). ISG15 impedes HIV-1 virus production by causing ISGylation of viral Gag protein and cellular factors, such as CHMP5 (chromatin-modifying protein 5) (46,47). APOBEC3G restricts HIV-1 replication by causing hypermutation of viral cDNA during reverse transcription (37,57).…”

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

“…These interactions allow LIP5 to coordinate ESCRT-III and VPS4 activities both during normal cellular homeostasis and also apparently in response to interferon signaling during innate immune responses. In the latter case, Leis and co-workers (63,64) have reported that covalent modification of CHMP5 and other ESCRT-III proteins with the ubiquitin-like protein ISG15 blocks the LIP5-CHMP5 and LIP5-VPS4 interactions and thereby inhibits enveloped viruses from using the ESCRT pathway to bud from cells. To define better how LIP5 can mediate its different regulatory functions, we have characterized how LIP5 interacts with CHMP5 and other ESCRT-III proteins and determined the interdependence of different LIP5-ligand interactions both in vitro and in cells.…”

Mechanism of Inhibition of Retrovirus Release from Cells by Interferon-Induced Gene ISG15

“…Several reports show that ISG15 expression inhibits the E3 subunit of the ligase involved in Gag or Tsg101 ubiquitination, suggesting a possible mechanism for the observed inhibition (14,19,20,44). Disruption of the late release process is a more general antiviral mechanism in which the VPS4 ATPase is removed from the budding complex (25). When ISG15 and its ligase are expressed in cells, CHMP5 becomes ISGylated and accumulates in the membrane fraction (25).…”

“…A cellular innate immunity mechanism which targets retrovirus budding both early and late in the release process induces the expression of interferon-stimulated gene 15 (ISG15) and its specific ISG15 E1, E2, and E3 ligase complex (3,4,25,27,33,43,45). ISG15 is a dimer homologue of ubiquitin.…”