Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells

Abstract

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-like family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominantnegative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.RSV ͉ Gag ͉ virus particle assembly O ne of the least understood aspects of viral replication is the assembly of virions and their exit from the host cell. However, it has been demonstrated that determinants of retroviral assembly and budding lie solely within the Gag polyprotein (1, 2). Mutational analyses of Gag revealed three distinct domains involved in virion assembly: a membrane-binding domain (M domain), found at the amino terminus of matrix (MA), which contains a myristoylation signal in most retroviruses (1, 3, 4); an internal Gag interaction domain (I domain), which maps to nucleocapsid (NC) in RSV (5), and is involved in the aggregation of Gag polyproteins necessary for particle formation; and a late assembly domain (L domain), required late in the budding process (6-10).For Gag particle budding, a functional L domain is not required on every Gag molecule (9); moreover, the L domain can function at other positions in Gag, suggesting that it is a protein interaction domain (10). The RSV Gag L domain maps specifically to a proline-rich sequence of p2b, PPPPYV, located between the MA and capsid (CA) proteins in Gag (9, 10). Referred to as the PY motif, this sequence is found in the Gag protein of many retroviruses, but not lentiviruses, and in numerous cellular proteins. Interestingly, this motif is also found in the structural proteins of other budding viruses, including rhabdoviruses (vesicular stomatitis virus) and filoviruses (Ebola and Marburg viruses; ref. 11). For VSV, this PY motif has recently been shown to function in the late steps of budding, analogous to the L domain of the retroviral Gag (12). The PY motif specifically binds to a protein-interaction domain found mostly in signaling and regula...

PPPYEPTAP Motif Is the Late Domain of Human T-Cell Leukemia Virus Type 1 Gag and Mediates Its Functional Interaction with Cellular Proteins Nedd4 and Tsg101

“…The WW domains in Nedd4 bind PPPY motif in HTLV-1, as has been reported for RSV and MPMV retroviruses (26,48,66) and Ebola virus (17,18,33,60). Consequently, a subpopulation of retroviral Gag (ϳ2%) and VP40 is believed to become monoubiquitinated (17,18,47,57,58).…”

“…Depletion of Tsg101 by RNAi from the cytoplasm of mammalian cells (13) or overexpression of the N-terminal UEV domain of Tsg101 (9,14) arrested or decreased the release of HIV-1, functionally linking Tsg101 to virus budding and release. E3 ubiquitin ligases of the Nedd4 family were shown to be involved in budding of RSV and MPMV retroviruses (26,48,66). Proteasome inhibitors were found to interfere with the release of HIV-1 (55) and RSV (47), suggesting that depletion of free ubiquitin from the cytoplasm of mammalian cells disrupts retroviral budding and release.…”

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases

“…The HIV-1 Gag polyprotein is ubiquitylated, and while viral budding is abrogated in the absence of a functional ubiquitylation system, it remains unclear whether this relates to ubiquitylation of Gag or of the endocytic machinery. Further, Gag polyproteins of some viruses utilize a PTAP late domain motif to bind to the UEV domain of Tsg101, while Gag proteins of other viruses bind to a Nedd4 family E3 ligase (Kikonyogo et al, 2001). Binding of Gag HIV protein to Tsg101 induces its translocation from endosomes to the plasma membrane.…”

Differential Activities of Glucocorticoid-induced Leucine Zipper Protein Isoforms

“…Cells were then transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen) and optimized DNA concentrations (200 ng each of ␣-, ␤-, and ␥-FLAG-xENaC and 250 ng of various GILZ constructs/well). After treatment, the coverslips were removed and processed to detect expression of cell surface ENaC using live cell staining or using permeabilized staining to detect total cellular ENaC, as described previously (18,25). Cell surface ENaC was visualized using FLAG monoclonal antibody M2 (Sigma) and goat antimouse secondary antibody conjugated to Alexa-594 fluorophore (Molecular Probes, Inc., Eugene, OR) followed by nuclear co-staining with 4Ј,6-diamidino-2-phenylindole.…”