Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins - PubMed (original) (raw)
Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins
Juan Martin-Serrano et al. Proc Natl Acad Sci U S A. 2003.
Erratum in
- Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):152845. Yaravoy, A [corrected to Yarovoy, A]
Abstract
The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein-protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.
Figures
Fig. 1.
Interactions between mammalian class E VPS proteins. (A) Matrix showing interactions between VPS proteins in the yeast two-hybrid assay. Shading indicates the level of β-galactosidase activity in optical density (OD) units as indicated by the key. The GAL4-fusions of VPS28, CHMP5, and Hrs were strong transcriptional activators in the absence of a coexpressed VP16-fusion protein and were excluded. A more detailed version of these data are given in Table 1. (B) Examples of coprecipitation assays to evaluate class E VPS protein interactions. GST fusion proteins were coexpressed with one of a series of YFP fusion proteins, as indicated. Samples of clarified cell lysate and glutathione-Sepharose bead-bound proteins were analyzed by Western blot with an α-GFP antibody. In each case, the full-length ORF was fused to YFP, except in the Lower Right, where a series of truncated Tsg101 proteins were also used. (C) Interpretation of the yeast two-hybrid assay and coprecipitation results in the form of a protein–protein interaction map incorporating components of ESCRT-I, ESCRT-II (gray shading), ESCRT-III (black shading), and additional human homologues of yeast class E genes. Positive interactions scored by yeast two-hybrid assay are indicated by solid lines. Interactions observed in the GST fusion coprecipitation assay, but not in the yeast two-hybrid assay, are indicated by dotted lines.
Fig. 2.
Generalized L-domain dysfunction induced by ESCRT-III perturbation. (A) Infectious HIV-1 release mediated by HIV-1 p6 or EIAV p9 L domains in the complementation assay in the presence of YFP class E VPS fusion protein expression. Infectious virion production, measured by β-galactosidase assay after infection of P4/R5 cells, is plotted as a percentage of that obtained in the presence of unfused YFP (371,390 relative light units for HIV-p6 and 79,938 relative light units for HIV-p9). (B) Gag processing and virion release defects induced by inhibitory YFP-CHMP fusion protein expression. (C) Electron microscopic images of late viral budding defects induced by YFP-CHMP fusion proteins. The top six pairs of images show HIV-1 budding in the absence or presence of the inhibitory YFP-CHMP fusion proteins, whereas the bottom pair of images shows MLV budding in the presence of YFP-CHMP2A.
Fig. 3.
YPDL-dependent EIAV p9 binding to AIP-1/ALIX. (A) Yeast two-hybrid analysis. β-galactosidase reporter levels in yeast expressing GAL4 or GAL4-AIP-1/ALIX and VP16 or VP16-EIAV p9 were plotted. The p9 (M) mutant contained a four-residue substitution (YPDL to AAAA). (B) GST fusion proteins were expressed in 293T cells along with HA-tagged AIP-1/ALIX. Samples of clarified cell lysate and glutathione-bound proteins were analyzed by Western blot. Equivalent loading of the GST fusion proteins was verified by Coomassie blue staining of the glutathione-bound fraction.
Fig. 4.
A truncated form of AIP-1/ALIX that lacks ESCRT-III-binding activity is a specific inhibitor of a YPDL-type L domain. (A) AIP-1/ALIX domain organization. The indicated truncated versions of AIP-1/ALIX were tested for interaction with Tsg101, AIP-1/ALIX, CHMP4A, -B, -C, and EIAV p9 in the yeast two-hybrid assay. The ± ascribed to p9 interaction with AIP-1/ALIX residues 1–503 indicates a weak positive (β-galactosidase reporter levels ≈10% of those obtained by using the full-length or 503–869 residue fragment). (B) Western blot analysis of cell lysates and pelleted virions after transfection of 293T cells with a p6-deleted HIV-1 proviral plasmid, along with complementing Gagδp6-p6, Gagδp6-p9, and Gagδp6-PTAP expression vectors (Left). Alternatively, EIAV vector plasmids were transfected (Right). YFP or YFP-δ1–176 AIP-1/ALIX was coexpressed, as indicated. (C) Results are the same as for B, except that HIV-1 in culture supernatants was quantified by infection of P4/R5 indicator cells and β-galactosidase activity measurement, given in relative light units (RLU), or infectious EIAV virions were measured by titration on CRFK cells given in infectious units (iu)/ml of supernatant.
Fig. 5.
Effects of siRNA-mediated AIP-1/ALIX depletion on YPDL and PTAP-type L-domain function. (A) Validation of AIP-1/ALIX siRNAs. The 293T cells transfected with YFP or YFP-AIP-1/ALIX expression plasmids in the presence of siRNAs directed against luciferase (control) or AIP-1/ALIX, as indicated. Bright-field and fluorescent images of the same field, acquired 24 h after transfection, are shown. (B) Inhibition of virus release by Tsg101- and AIP-1/ALIX-specific siRNAs. Gagδp6-p6-, Gagδpb-p9-, and Gagδpd-PTAP-complemented HIV-1 was generated as in Fig. 4, but, in this case, luciferase (control)-, Tsg101-, or AIP-1/ALIX-specific siRNAs were cotransfected. Virion production was measured by using P4/R5 cells as in Fig. 4. (C) Western analysis of virion HIV-1 formation mediated by p6, p9, or PTAP in the presence of siRNAs. Lanes L, luciferase control siRNAs; lanes A, AIP-1/ALIX siRNAs.
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