Multiple interactions between the ESCRT machinery and arrestin-related proteins: implications for PPXY-dependent budding

Abstract

Late domains are short peptide sequences encoded by enveloped viruses to promote the final separation of the nascent virus from the infected cell. These amino acid motifs facilitate viral egress by interacting with components of the ESCRT (endosomal sorting complex required for transport) machinery, ultimately leading to membrane scission by recruiting ESCRT-III to the site of viral budding. PPXY late (L) domains present in viruses such as murine leukemia virus (MLV) or human T-cell leukemia virus type 1 (HTLV-1) access the ESCRT pathway via interaction with HECT ubiquitin ligases (WWP1, WWP2, and Itch). However, the mechanism of ESCRT-III recruitment in this context remains elusive. In this study, we tested the arrestin-related trafficking (ART) proteins, namely, ARRDC1 (arrestin domain-containing protein 1) to ARRDC4 and TXNIP (thioredoxin-interacting protein), for their ability to function as adaptors between HECT ubiquitin ligases and the core ESCRT machinery in PPXY-dependent budding. We present several lines of evidence in support of such a role: ARTs interact with HECT ubiquitin ligases, and they also exhibit multiple interactions with components of the ESCRT pathway, namely, ALIX and Tsg101, and perhaps with an as yet unidentified factor. Additionally, the ARTs can be recruited to the site of viral budding, and their overexpression results in a PPXY-specific inhibition of MLV budding. Lastly, we show that WWP1 changes the ubiquitination status of ARRDC1, suggesting that the ARTs may provide a platform for ubiquitination in PPXY-dependent budding. Taken together, our results support a model whereby ARTs are involved in PPXY-mediated budding by interacting with HECT ubiquitin ligases and providing several alternative routes for ESCRT-III recruitment.

FIG. 1.

(A) Schematic drawing of human ARTs featuring N- and C-terminal arrestin-like domains as well as PPXY and PSAP motifs in the C terminus as indicated. (B) Yeast two-hybrid screen for interactions of ART proteins with HECT ubiquitin ligases, components of the ESCRT machinery, and ubiquitin. ARTs were expressed as fusion constructs to the Vp16 activation domain for screening against WWP1, Nedd4, and Itch or fused to the GAL4 DNA binding domain for testing binding to WWP2, Tsg101, ALIX, and ubiquitin. Protein interactions were determined by measuring β-galactosidase activity levels and are shown as absorbance units (optical density at 540 nm [OD 540]). Values above saturation levels were arbitrarily set to 5. Error bars indicate the standard deviations from the mean of two independent experiments measured in triplicates.

FIG. 2.

(A) Coprecipitation assays showing interactions of ARRDC1, -2, and -3 with WWP1 (WW domains) (left), of ARRDC1 and -2 with Tsg101 (middle), and of ARRDC1 with ALIX (right). Proteins fused to GST, HA, or YFP were transiently expressed in 293T cells as indicated, and GST-tagged proteins were precipitated using glutathione-Sepharose beads. Samples were analyzed for coprecipitated binding partners by SDS-PAGE followed by Western blotting (WB). Figures shown are representative of at least two independent experiments. α, anti. (B) Colocalization of ALIX and Tsg101 with ARRD1. Proteins fused to mCherry, YFP, or CFP as indicated were transiently expressed in 293T cells, and fixed cells were analyzed for protein colocalization by confocal microscopy. Pictures shown are representative of at least two independent experiments. Colocalization of coexpressed proteins was quantified by calculating the Pearson's correlation coefficient using Image J public domain image processing software (plug-in). This analysis yielded the following results: mCherry-ARRDC1/YFP, −0.07 ± 0.18 (n = 7); mCherry/YFP-Tsg101, 0.10 ± 0.07 (n = 8); mCherry-ARRDC1/YFP-Tsg101, 0.7 ± 0.18 (n = 11); YFP-ARRDC1/CFP, −0.26 ± 0.16(n = 12); YFP/CFP-ALIX, 0.55 ± 0.11 (n = 7); YFP-ARRDC1/CFP-ALIX, 0.69 ± 0.11 (n = 9). Data are represented as average value ± standard deviation. n, number of analyzed cells.

FIG. 3.

(A) Schematic drawing of ARRDC1 wild type (ARRDC1) and truncation mutant (ARRDC1NC) lacking the C terminus. (B) Binding of ARRDC1 to HECT ubiquitin ligases, ALIX, and Tsg101 is dependent on binding motifs in the C terminus. C-terminally truncated ARRDC1 fused to the Vp16 activation domain or GAL4 DNA binding domain was tested for its ability to interact with WWP1, WWP2, Nedd4, Itch, ALIX, and Tsg101 by yeast two-hybrid screening. Protein interactions were detected by measuring β-galactosidase activity in yeast lysates at an optical density at 540 nm (OD 540). Error bars indicate the standard deviations from the mean of two independent experiments measured in triplicates. (C) Expression of full-length (fl) and C-terminally truncated (NC) ARRDC1, ARRDC2, and ARRDC3 in yeast lysates. ARTs were expressed in the context of the HB18 vector (Vp16 activation domain), thereby generating an HA fusion protein. Protein expression was determined by lysis of yeast in protein sample buffer, followed by SDS-PAGE and Western blotting (WB) against HA. α, anti. (D) Schematic drawing of ALIX featuring the Bro1 and the V domain as well as the proline-rich region (PRR). (E) Binding of ARRDC1, ARRDC2, and ARRDC3 to ALIX is dependent on the proline-rich region in Alix. ARTs, CHMP4B, and Cin85 expressed as fusion proteins to the GAL4 DNA binding domain were tested for binding to ALIX by yeast two-hybrid screening as described for panel A.

FIG. 4.

ARRDC1 can be ubiquitinated by WWP1. 293T cells were transiently transfected with expression constructs for HA-ubiquitin, GST, GST-ARRDC1, or GST-ARRDC1NC together with YFP constructs as indicated. GST-tagged proteins were subsequently isolated using glutathione-Sepharose beads, and samples were analyzed by Western blotting (WB). Western blots were performed using an infrared imaging system that allowed detection of total amounts of ARRDC1 (left panel) as well as its ubiquitinated forms using anti-HA antibodies (middle panel) on the same membrane. An overlay picture of signals acquired in both channels is shown in the right panel. Results presented are representative of three independent experiments. α, anti.

FIG. 5.

Ebola virus VP40 can recruit ARRDC1 to the plasma membrane, depending on its PTAPPEY motif. 293T cells were transiently transfected with expression constructs for mCherry-ARRDC1 and YFP-WWP1 featuring a deletion of its membrane-targeting domain (YFP-WWP1ΔC2). Localization of ARRDC1 and WWP1ΔC2 upon expression of either empty vector (pCR3.1), wild-type Ebola virus matrix protein VP40 (myc-EbVP40), or a VP40 deletion mutant lacking the late domain (myc-EbVP40δ1-13) was analyzed by confocal microscopy after cells were fixed and nuclei were stained with Hoechst. Localization of mCherry-ARRDC1 and CFP-WWP1ΔC2, either intracellular or at the plasma membrane, was determined by counting at least 50 cells for samples expressing wild-type or late-domain-deficient VP40 (graph). Pictures and graph shown are representative of two independent experiments.

FIG. 6.

PPXY-mediated viral particle production is specifically reduced upon transient expression of ART proteins. (A) Analysis of infectious particle production. 293T cells were transfected with MLV provirus expression vectors containing its natural PPXY L domain (MLV) or a mutant depending on a PTAP motif (MLV hPTAP) as well as expression constructs encoding YFP fusion proteins as indicated. Infectious virus release was analyzed using a chemiluminescent assay after infection of TZM-bl reporter cells with supernatants harvested from the transfected 293T cells. In these experiments, absolute infectivity values of 2.4 × 10⁶ for MLV and 1.0 × 10⁶ for MLV hPTAP were obtained on average for cells expressing unfused YFP, and these values were set to 100% to calculate relative infectious virion release for either MLV or MLV hPTAP. Error bars indicate the standard deviation from the mean of at least three independent experiments. (B) Analysis of virus-like particle release. Virus-like particles (VLPs) were isolated from 293T cells transiently expressing HA-tagged MLV Gag featuring either a PPXY (MLV) or a mutant PTAP L domain (MLV hPTAP) together with the indicated YFP fusion proteins. VLPs and cellular lysates were analyzed by Western blotting (WB) against the MLV HA tag; signal intensities were measured using Odyssey, version 3.0, software, and budding efficiency was determined. The depicted Western blots are representative of three independent experiments; the graph illustrates the budding efficiency gained upon expression of YFP-ARRDC1NC. Error bars indicate standard deviations from three independent experiments. α, anti.

FIG. 7.

Full-length as well as dominant negative ARRDC1 colocalizes with aberrant endosomes induced by dominant negative VPS4. HeLa cells were transiently transfected with expression constructs encoding mCherry-ARRDC1 or mCherry-ARRDC1NC and dominant negative YFP-VPS4 (YFP-VPS4dn). Cells were fixed, and nuclei were stained using Hoechst stain and analyzed by confocal microscopy for colocalization of ARRDC1 proteins with aberrant endosomes induced by the expression of dominant negative VPS4. Pictures shown are representative of three independent experiments. Colocalization of ARRDC1 constructs with dominant negative VPS4 was quantified by calculating the Pearson's correlation coefficient using Image J public domain image processing software (plug-in). The following values were obtained: mCherry-ARRDC1/YFP, 0.16 ± 0.04 (n = 3); mCherry-ARRDC1/YFP-VPS4dn, 0.66 ± 0.10 (n = 7); mCherry-ARRDC1NC/YFP, −0.24 ± 0.15 (n = 7); mCherry-ARRDC1NC/YFP-VPS4dn, 0.76 ± 0.15 (n = 5). Data are represented as average value ± standard deviation. n, number of analyzed cells.

FIG. 8.

Schematic drawing of three possible mechanisms of ESCRT-III recruitment by ART proteins during PPXY-mediated viral budding. ARTs might function by binding to both HECT ubiquitin ligases and components of the ESCRT machinery, i.e., ALIX and Tsg101, as well as an uncharacterized factor, thereby recruiting the ESCRT-III scission activity (left panel). Second, ubiquitination of ARTs induced by HECT ubiquitin ligases might recruit ESCRT-III via interactions of ESCRT-0, -I, and -II with ubiquitin moieties (middle panel). Third, binding of ARTs to ubiquitinated viral proteins might induce ESCRT-III recruitment via mechanisms described above (right panel). CA, capsid; NC, nucleocapsid.