JVI Accepts, published online ahead of print on 28 October 2009

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J. Virol. doi:10.1128/JVI.01319-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Late domain-independent rescue of a release-deficient Moloney Murine Leukemia Virus by the Ubiquitin ligase Itch

Joshua A. Jadwin, Victoria Rudd, Paola Sette, Swathi Challa, and Fadila Bouamr^*

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, 20892, MD

^* To whom correspondence should be addressed. Email: bouamrf{at}mail.nih.gov.

Moloney Murine Leukemia Virus (MoMLV) Gag utilizes its Late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell's budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that over-expression of Tsg101 or Alix failed to rescue the release of PPPY deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2 or Nedd4.2s did not rescue this release deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch, and an intact C2 domain, but not the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif, and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue and their absence did not affect the incorporation of Itch into the rescued particles. Itch mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA_i knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY lacking-MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in a L domain independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.