Rescue of HIV-1 Release by Targeting Widely Divergent NEDD4-Type Ubiquitin Ligases and Isolated Catalytic HECT Domains to Gag

Abstract

Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

Multiple Interactions between the ESCRT Machinery and Arrestin-Related Proteins: Implications for PPXY-Dependent Budding

“…However, this residue is conserved only in ARRDC1 and TXNIP while ARRDC2, ARRDC3, and ARRDC4 lack lysine residues in their C termini, suggesting that lysines in the NC region may be modified by ubiquitination in humans. It also remains to be addressed whether ART ubiquitination by WWP1 is K63 linked, a posttranslational modification that has been involved in MVB sorting (30) and retroviral budding (56,60). In fact, WWP1 is likely to synthesize K63-linked ubiquitin chains (29), suggesting that the ARTs might simply provide a platform for this type of ubiquitination by virtue of being at the sites of viral assembly.…”

“…For example, it is now established that Nedd4L can stimulate the release and infectivity of HIV-1 viruses that lack the PTAP and LYPXL late domains, and this activity requires the enzymatic activity encoded by Nedd4L (8,57,60). Additionally, Itch is recruited by MLV Gag in a PPXY-independent manner to promote viral egress, and, as shown for PPXY-dependent budding, this activity also requires the core ESCRT machinery (25).…”

“…The fact that the enzymatic activity of HECT ubiquitin ligases is critical for their function in viral budding (34,50,60) suggests two possible scenarios for ESCRT-III recruitment in PPXY-mediated budding: the target for ubiquitination by HECT ubiquitin ligases could be a…”

“…The fact that the enzymatic activity of HECT ubiquitin ligases is critical for their function in viral budding (34,50,60) suggests two possible scenarios for ESCRT-III recruitment in PPXY-mediated budding: the target for ubiquitination by HECT ubiquitin ligases could be a viral protein that would subsequently recruit ESCRT-III via interactions of ESCRT proteins with ubiquitin, or, alternatively, an unidentified ubiquitin acceptor could serve as a bridge between HECT ubiquitin ligases and the core ESCRT machinery. Accordingly, the observation that cumulative mutation of lysine residues in Gag can lead to a late budding arrest in HIV-1 (15) and Rous sarcoma virus (RSV) (52) suggested that Gag might be the relevant substrate for L-domain activity, a hypothesis that is consistent with the functional rescue of an L-domain-deficient Gag upon fusion to ubiquitin (26).…”

A new HECT ubiquitin ligase regulating chemotaxis and development in Dictyostelium discoideum

“…It is possible that, in the absence of the long N-terminal sequence, the overexpressed HECT wt domain binds E2 enzymes, transferring the ubiquitin moiety indiscriminately to specific substrates responsible for the HSB1 phenotype in addition to nonspecific substrates (Weiss et al, 2010;Park et al, 2009).…”