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Address correspondence to Wesley I. Sundquist, Dept. of Biochemistry, University of Utah, 20 N, 1900 E, Rm. 211, Salt Lake City, UT 84132. Tel.: (801) 595-8203. Fax: (801) 581-7959. email: wes{at}biochem.utah.edu
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Abstract |
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Key Words: virus budding; virions; ubiquitin; vacuolar protein sorting; multivesicular body
O. Pornillos' present address is The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037.
Abbreviations used in this paper: ESCRT, endosomal sorting complex required for transport; Hrs, hepatocyte growth factorregulated tyrosine kinase substrate; MVB, multivesicular body; Tsg101, tumor susceptibility gene 101; UEV, ubiquitin E2 variant; UIM, ubiquitin-interacting motif; VLP, virus-like particle; Vps, vacuolar protein sorting.
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Introduction |
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In the cell, Tsg101 (yeast Vps23p) normally functions as a subunit of the endosomal sorting complex required for transport-I (ESCRT-I) protein complex (Katzmann et al., 2001). Tsg101 and ESCRT-I perform essential roles in the vacuolar protein sorting (Vps) pathway, in which integral membrane proteins such as cell surface receptors are targeted for destruction in the lysosome (for review see Katzmann et al., 2002). In this pathway, monoubiquitylated proteins are delivered to endosomal membranes where they are sorted into microdomains that ultimately bud as small vesicles into late endosomal compartments to form multivesicular bodies (MVBs). MVBs can then fuse with lysosomes and release these vesicles and their protein cargos into the lumen of the lysosome, where they are degraded by hydrolases and lipases.
ESCRT-I is one of a series of soluble protein complexes that are recruited from the cytoplasm onto the surface of the endosome during MVB biogenesis. Once on the membrane, Tsg101/ESCRT-I appears to perform at least two essential functions: (1) it binds ubiquitylated cargo proteins via direct interactions between the Tsg101 UEV domain and the ubiquitin modifications (Katzmann et al., 2001); and (2) it helps to recruit the downstream ESCRT-II, ESCRT-III, Vps4, and other proteins and complexes required to complete protein sorting and vesicle formation (Babst et al., 2002a, b). Thus, recruitment of Tsg101 to the membrane surface represents a key step in the commitment to vesicle formation. However, the mechanism by which Tsg101/ESCRT-I is recruited to the membrane has not been elucidated.
Although interactions between Tsg101 and cellular P(S/T)AP motifs have not been described, we reasoned that such interactions might occur and play a role in the recruitment of Tsg101 to the endosomal membrane. Protein database searches revealed several Vps proteins with PTAP or PSAP motifs (Garrus et al., 2001), including (1) Tsg101 itself; and (2) hepatocyte growth factorregulated tyrosine kinase substrate (Hrs; yeast Vps27p; Raiborg et al., 2001a; Raiborg and Stenmark, 2002). Hrs is an attractive candidate for the factor that recruits Tsg101 to endosomal membranes because it is required for receptor down-regulation and MVB biogenesis (Piper et al., 1995; Odorizzi et al., 1998; Lloyd et al., 2002; Shih et al., 2002; Bache et al., 2003b) and because it interacts with ubiquitylated cargo proteins on the early endosome (and therefore appears to function upstream of Tsg101; Bilodeau et al., 2002; Bishop et al., 2002; Raiborg et al., 2002; Bache et al., 2003b).
The domain organization and biochemical properties of the Hrs and HIV-1 Gag proteins exhibit several intriguing similarities (Fig. 1). First, both proteins contain amino-proximal membrane-targeting domains. In HIV-1 Gag, the N-myristoylated MA domain binds membranes, and contains signals that target Gag to the plasma membrane in some cell lines (Göttlinger, 2001; Raposo et al., 2002; Pelchen-Matthews et al., 2003). In Hrs, the FYVE domain binds PI(3)P and thereby targets Hrs to endosomal membranes (Komada et al., 1997; Odorizzi et al., 1998; Raiborg et al., 2001b; Katzmann et al., 2003). Second, both proteins contain P(S/T)AP motifs within proline-rich (and apparently unstructured) regions. Third, both Hrs and HIV-1 Gag proteins are monoubiquitylated. Although a functional role for HIV Gag ubiquitylation in retrovirus budding has not been demonstrated, ubiquitin transfer is important for virus budding (for review see Vogt, 2000), and viral late domains have been shown to recruit ubiquitin ligase activities (Strack et al., 2000, 2002). Hrs ubiquitylation is essential for receptor down-regulation through the MVB pathway, and requires a cis-acting sequence motif called the ubiquitin-interacting motif (UIM; Young et al., 1998; Hofmann and Falquet, 2001; Bishop et al., 2002; Lloyd et al., 2002; Polo et al., 2002; Raiborg et al., 2002; Shih et al., 2002). Other UIM-containing proteins are substrates for the Nedd4 (yeast Rsp5p) family of ubiquitin ligases (Katz et al., 2002; Polo et al., 2002), and although it is not yet known whether Nedd4 ubiquitylates Hrs, the protein does harbor a PPEY motif. This sequence matches the consensus sites for cellular Nedd4 substrates and for PPXY viral late domains (Rotin et al., 2000; Freed, 2002; Pornillos et al., 2002c).
The preceding observations are consistent with a model in which (1) Tsg101 is normally recruited to the endosomal membrane through a direct interaction with Hrs; and (2) the HIV-1 Gag protein has evolved to mimic the Tsg101-recruiting functions of Hrs, and thereby redirect the machinery of MVB vesicle formation to sites of viral budding on the plasma membrane. Experiments described in this paper were designed to test key aspects of these models for Hrs function and viral mimicry.
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Results |
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Interaction of full-length Tsg101 and Hrs proteins
The interaction of full-length Tsg101 and wild-type Hrs proteins was examined in directed yeast two-hybrid assays. Plasmids expressing a Tsg101Gal4p binding domain fusion (Tsg101-BD) and an HrsGal4p activation domain fusion (Hrs-AD) were cotransfected into the reporter yeast strain J693, resulting in significant levels of ß-galactosidase activity (Fig. 3 A, top left). In contrast, control experiments in which one of the two plasmids encoded only the Gal4p BD or AD alone did not produce significant ß-galactosidase activity (Fig. 3 A). Similarly, in semi-quantitative liquid culture assays, the binding of full-length Hrs-AD to full-length Tsg101-BD typically stimulated ß-galactosidase activity 100-fold above background (Fig. 3, BD). Therefore, we conclude that the full-length Tsg101 and Hrs proteins interact in the yeast two-hybrid assay.
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The ubiquitin-binding mutation in Tsg101 UEV (N45A) had no effect on the Hrs/Tsg101 interaction (Fig. 3 B). Likewise, alanine substitutions in the Hrs PPEY sequence had no significant effect on Tsg101/Hrs binding (unpublished data). These experiments indicated that the Tsg101/Hrs two-hybrid interaction was not mediated through ubiquitin.
In contrast, the PSAP-binding mutation in Tsg101 (M95A) reduced Hrs binding significantly (approximately sixfold; Fig. 3 C). In complementary experiments, alanine substitution of the Hrs 348PSAP351 sequence (HrsPSAP) also reduced wild-type Tsg101 binding, albeit to a lesser extent (approximately twofold). The greater reduction in binding seen for the Tsg101 M95A mutation may reflect weak Tsg101 UEV binding to several additional PSAP-like motifs (583PSGP586 and 620PSMP623) that are conserved from human Hrs to yeast Vps27p (Emr, S., personal communication). As expected, the Hrs
PSAP mutation did not further reduce binding of the Tsg101 M95A mutation, consistent with the idea that these mutations affected the two sides of the same proteinprotein interface.
Surprisingly, even in the absence of the Tsg101 UEV/Hrs PSAP interaction, Hrs and Tsg101 exhibited significant residual binding. Therefore, directed two-hybrid experiments were used to define the complete Hrs region required for full affinity binding. As summarized in Fig. 3 D, Tsg101 did not bind the NH2-terminal VHS and FYVE domains of Hrs (residues 1221). These domains were also completely dispensable for the Tsg101/Hrs interaction, as Tsg101 bound equally well to a COOH-terminal fragment spanning residues 222777 (HrsN) as to full-length Hrs. In contrast, deletion of COOH-terminal Hrs residues 565777 reduced (but did not eliminate) Hrs binding, and a further six-amino acid deletion that removed the entire COOH-terminal Pro/Gln-rich region of Hrs (Hrs
N
C; Hrs residues 222559) reduced Tsg101 binding to nearly background levels. A murine Hrs construct (residues 287573) that extended
15 residues into the Pro/Gln-rich region bound well (Bache et al., 2003a), and we therefore conclude that Hrs residues 560573 contribute to Tsg101 binding. Tsg101 bound only weakly (or not at all) to a series of smaller Hrs fragments that spanned just the Hrs PSAP element (e.g., Hrs 1450) or the coiled-coil Pro/Gln-rich region (e.g., Hrs 451777; see Fig. 3 D). Thus, we conclude that full affinity Tsg101 binding requires the presence of two (or more) different Hrs regions: (1) the PSAP motif; and (2) a second region spanning the putative coiled-coil and at least part of the Pro/Gln-rich region.
Hrs late domain activity
Next, we tested whether the protein-recruiting functions of Hrs were sufficient to support the budding of VLPs from cultured human cells. Expression of HIV GagGFP fusion proteins in human cell lines recapitulates many aspects of viral assembly and budding (Hermida-Matsumoto and Resh, 2000), including the requirement for Tsg101 and other components of the MVB pathway (Garrus et al., 2001).
Human embryonic kidney 293T cells were transfected with HIV Gag expression constructs, and analyzed for protein expression and for VLP release 2428 h later (Fig. 4). VLP release was analyzed by Western blotting of culture supernatants after sucrose cushion pelleting (Fig. 4, B and C; VLP). Intracellular Gag protein expression levels were analyzed in Western blots of cytoplasmic extracts (Fig. 4, B and C; Cell). As expected, the control GagGFP fusion protein expressed well and formed VLPs efficiently (Fig. 4 B, lane 1; Hermida-Matsumoto and Resh, 2000; Garrus et al., 2001). VLP release was dependent on the presence of a functional PTAP late domain, as mutation of the PTAP motif to LIRL (GagPTAPGFP; Fig. 4 B, lane 2) severely attenuated particle release (Göttlinger et al., 1991; Huang et al., 1995; Garrus et al., 2001).
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Analogous results were obtained in experiments designed to test whether GagHrs fusion proteins could rescue the budding of defective Gag constructs in trans (Fig. 4 C). This experiment took advantage of the fact that defective Gag proteins can be released from cells by co-assembling with Gag proteins that are competent for budding (Martin-Serrano et al., 2001). Control experiments demonstrated that wild-type GagGFP could rescue the release of Gagp6 when the two proteins were coexpressed in the same cell (Fig. 4 C, top, compare lane 1 and lane 2), and that rescue required the GagGFP PTAP sequence (Fig. 4 C, top, compare lane 2 and 3). Gag
p6 release was also efficiently rescued in trans by coexpression with Gag
PTAPHrs
N (Fig. 4 C, top, lane 4). Hence, we conclude that the Hrs
N polypeptide has potent late domain activity and can support HIV-1 Gag budding both in cis and in trans.
In addition to rescuing VLP formation in trans, some Gag fusion proteins with late domain activities can also rescue the infectivity of HIV-1 proviral constructs with budding defects (Martin-Serrano et al., 2001). For example, the wild-type GagGFP protein can rescue both the budding and infectivity of HIV-1 proviral constructs that harbor mutations in the PTAP-coding region of the Gag gene. However, the GagPTAPHrs
N construct did not efficiently rescue infectivity of the R9
PTAP proviral construct in a single cycle replication assay (unpublished data), and we speculate that this may reflect alterations in particle morphology caused by incorporation of the Hrs222777 polypeptide (see below).
Sequence requirements for Hrs late domain activity
To test the sequence requirements for Hrs late domain activity, HrsN constructs missing either the PSAP motif (Gag
PTAPHrs
N
PSAP) or the Pro/Gln-rich region (Gag
PTAPHrs
N
C) were also tested in the two VLP release assays. As shown in Fig. 4 B, Gag
PTAPHrs
N
C was not released from cells (Fig. 4 B, lane 5), whereas Gag
PTAPHrs
N
PSAP was released (Fig. 4 B, lane 4), but with reduced efficiency as compared with the Gag
PTAPHrs
N control (27 vs. 51% total Gag protein released). Similarly, Gag
p6 release was not efficiently rescued in trans by the Gag
PTAPHrs
N
C protein (Fig. 4 C, lane 6), although in some repetitions of this experiment, Gag
p6 was released at low levels (unpublished data). The low levels of release, when seen, presumably reflected weak residual late domain activity of the PSAP sequence within the Hrs
N
C polypeptide. The Gag
PTAPHrs
N
PSAP protein again rescued the release of Gag
p6 to some degree (Fig. 4 C, lane 5), but was again less efficient than Gag
PTAPHrs
N (Fig. 4 C, compare lane 4 and lane 5). Also, we tested the release of a Gag
PTAPHrs
N construct with a mutation in the UIM (265LA266
AL). This mutation abolishes Hrs ubiquitin binding and ubiquitylation of Hrs itself (Polo et al., 2002), but did not diminish VLP release. Indeed, the 265LA266
AL mutation actually appeared to enhance Gag
p6Hrs
N release slightly (unpublished data). Overall, we conclude that full Hrs late domain activity requires the 348PSAP351 element and COOH-terminal coiled-coil Pro/Gln-rich region, but not the UIM. These requirements correlate well with the requirements for Tsg101 binding.
EM analyses of VLPs
Transmission EM was used to confirm that the GagPTAPHrs
N protein was released in the form of VLPs and to examine the phenotypes of the Gag constructs that failed to bud efficiently. In control experiments, GagGFP VLPs appeared as enveloped, spherical particles that generally resembled immature HIV virions in both appearance and size (100200 nm in diameter; Fig. 5 A). However, the GFP polypeptide appeared to create discontinuities in the GagGFP layer, in contrast to the evenly distributed ring of Gag density that is normally observed beneath the membranes of immature HIV-1 virions and Gag VLPs.
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EM images of thin-sectioned cells were also examined to determine the phenotypes of the Gag fusion proteins that were not released efficiently (Fig. 5, CE). In cells expressing GagPTAPGFP, Gag
PTAPHrs
N
C, and Gag
PTAPHrs
N
PSAP, clusters of enveloped spherical particles associated with the cell surface were frequently observed (Fig. 5, CE; top images). Although it can be difficult to establish membrane connectivities in thin-sectioned images, many of the assembling VLPs clearly remained tethered to the plasma membrane via thin membrane stalks (Fig. 5, CE, arrows in bottom images). Also, we observed images in which it appeared that vacuolar structures filled with assembled VLPs were in the process of (or had recently) fused with the plasma membrane (unpublished data). These images suggested that at least some of the VLPs budded intracellularly, in good agreement with recent experiments showing that HIV-1 can bud into MVB compartments in macrophages (Raposo et al., 2002; Pelchen Matthews et al., 2003).
Overall, our EM analyses established that although the GagPTAPGFP, Gag
PTAPHrs
N
C, and Gag
PTAPHrs
N
PSAP proteins were not released from cells efficiently, they did associate with membranes and initiate spherical particle assembly. Thus, the block to particle release in these constructs occurred at a late stage in the budding process, and therefore reflected the defect(s) in late domain activity.
The late domain activity of Hrs is dependent on Tsg101
Next, we examined whether the late domain activity of the HrsN polypeptide required the presence of Tsg101. Cellular Tsg101 can be efficiently depleted using RNA interference (Garrus et al., 2001; Fig. 6, bottom, compare lane 1 and lane 2). As shown in Fig. 6, release of Gag
p6Hrs
N VLPs from 293T cells was blocked when Tsg101 was depleted from the producer cells (Fig. 6, top, compare lane 1 and lane 2). VLP release was restored when an exogenous RNA interferenceresistant Tsg101FLAG protein (denoted Tsg101*) was expressed (Fig. 6, compare lane 2 and lane 3). Therefore, this experiment confirmed that the late domain activity of Hrs
N is dependent on Tsg101.
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Discussion |
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Sequential recruitment of proteins onto the endosomal membrane
An intriguing aspect of MVB biogenesis is that a series of soluble complexes must be sequentially recruited from the cytoplasm onto subdomains of the endosomal membrane where ubiquitylated protein cargos accumulate and are eventually sorted into MVB vesicles. Hrs is the first of the soluble MVB factors to bind, and its recruitment is mediated, at least in part, by FYVE domain binding to PI(3)P molecules displayed on the endosomal membrane (Odorizzi et al., 1998; Urbe et al., 2000; Raiborg et al., 2001b; Katzmann et al., 2003). Hrs localization may also be cargo-dependent (e.g., through interactions between the Hrs UIM motif and the ubiquitin modifications on cargo proteins; Raiborg et al., 2002). Finally, interactions with other endosomal proteins may also play important roles in Hrs localization (Urbe et al., 2000; Raiborg et al., 2001b; Bache et al., 2002).
On the membrane, Hrs is an essential subunit of a multiprotein complex that includes Hrs, ubiquitylated protein cargos, STAM1, STAM2, Eps15, and clathrin (Asao et al., 1997; Raiborg et al., 2002; Yamada et al., 2002; Bache et al., 2003b). This complex next recruits the soluble Tsg101/ESCRT-I complex, and our experiments indicate that this occurs, at least in part, through a direct binding interaction between Hrs and Tsg101. The nature of the "switch" that allows Tsg101 recruitment only after Hrs is bound to the membrane is still unclear, but plausible possibilities include (1) avidity effects (as multiple copies of Hrs assemble at the membrane, and Tsg101 can also oligomerize; Martin-Serrano et al., 2003); (2) cooperative binding of Tsg101 to Hrs and other endosomal membrane protein(s) (e.g., ubiquitylated protein cargos); and/or (3) membrane-dependent Hrs conformational changes or phosphorylation events (Urbe et al., 2000; Bache et al., 2002).
In the next stages of MVB biogenesis, the soluble ESCRT-II and -III proteins are recruited from the cytoplasm onto the membrane (Babst et al., 2002b). Again, it seems likely that the membrane-bound ESCRT-I must be "activated" in some fashion to allow the recruitment of these downstream factors only after ESCRT-I is bound to the membrane.
Possible mechanisms of Tsg101 recruitment and activation
Unexpectedly, our binding experiments suggested that full affinity Tsg101 binding requires at least two Hrs elements, a first which involves the Hrs PSAP sequence binding to the Tsg101 UEV domain, and a second that includes downstream Hrs elements that spans the putative coiled-coil region and at least part of the Pro/Gln-rich region. Both interactions were functionally important, although mutations that disrupted the Tsg101 UEV/PSAP interaction did not completely eliminate complex formation and VLP budding in all contexts, indicating that the downstream Hrs elements may play the dominant role in Tsg101 recruitment.
The use of multiple contact sites could simply serve to increase the affinity and specificity of the Hrs/Tsg101 interaction. However, an attractive alternative model is that the downstream Hrs site serves primarily to recruit Tsg101 to sites of vesicle budding, whereas the Hrs PSAP element serves primarily as the switch that "activates" Tsg101 for ESCRT-II and/or MVB cargo binding (or for other essential events in MVB biogenesis). In this model, we envision that the soluble cytoplasmic ESCRT-I protein may exist primarily in an autoinhibited conformation in which the Tsg101 UEV domain binds its own PTAP element (i.e., in cis; Fig. 7 A). Hrs binding could then drive a conformational change in which the Tsg101 UEV domain switches to bind the PSAP element of Hrs (i.e., in trans). This activation event might also involve the ubiquitin-binding site on the UEV domain interacting with ubiquitin modifications on protein cargos, which would provide a cooperative binding mechanism for insuring that activation occurs only in the presence of both Hrs and ubiquitylated protein cargos. Although this model remains to be tested rigorously, we note that analogous autoinhibition/conformational switching mechanisms are used by other classes of proteins, such as protein kinases, to create directionality in other cellular pathways (Francis et al., 2002; Pellicena and Miller, 2002).
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Materials and methods |
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All Gag and GagHrs constructs were based on pGagGFP, which contains the Rev-independent HIV-1HBX2 Gag sequence fused to EGFP (Hermida-Matsumoto and Resh, 2000; a gift from Marilyn Resh, Sloan-Kettering Institute, New York, NY). GagPTAPGFP was created by mutating the Gag PTAP motif to LIRL as described previously (Garrus et al., 2001). Gag
PTAPHrs fusion constructs were created from the Gag
PTAPGFP construct by replacing the last three amino acids of Gag and the GFP-coding region with the appropriate Hrs sequences. Gag
p6 and Gag
p6Hrs constructs were created by replacing the p6- and GFP-coding regions with either a stop codon or the appropriate Hrs sequences. Hrs constructs were PCR amplified from an EST (GenBank/EMBL/DDBJ accession no. BE276844; from American Type Culture Collection, Manassas, VA).
For yeast two-hybrid assays, Tsg101 constructs were PCR amplified from pIRES-TsgFlag and subcloned into the plasmid MP30 (Myriad Genetics), which encodes the Gal4p DNA-binding domain (BD). Hrs constructs were subcloned into the plasmid MP29 (Myriad Genetics). The resulting plasmids encoded in-frame fusions of Tsg101 with the Gal4p BD and of Hrs with the Gal4p activation domain (AD). All constructs were verified by DNA sequencing. Full cloning details are available from the authors.
Biosensor binding assays
BIAcore biosensor measurements of Tsg101 UEV binding to immobilized GSTpeptide fusion constructs were performed as described previously (Garrus et al., 2001), and purified recombinant Tsg101 UEV domain and GSTpeptide fusion constructs were also obtained as described previously (Jenkins et al., 2001; Pornillos et al., 2002a).
Yeast two-hybrid binding assays
Yeast strain J693 (MAT ade2 his3 leu2 trp1 cyh2 ura3::GAL1p-LacZ gal4 gal80 lys2::GAL1p-HIS3; Bendixen et al., 1994) was cotransformed with various pairs of BD and AD constructs (using empty BD and AD vectors as controls), and plated on synthetic media lacking the appropriate amino acids. To identify colonies producing ß-galactosidase, cells were lifted onto filter paper disks, lysed by freeze-thawing in liquid nitrogen, and assayed with X-gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside). For quantitation of ß-galactosidase activity, colonies (
20 per plate) were selected at random, pooled, grown on synthetic liquid media, and assayed in triplicate with CPRG (chlorophenol red ß-D-galactopyranoside) or ONPG (o-nitrophenyl-ß-D-galactopyranoside) as described previously (Garrus et al., 2001). These experiments were repeated a total of six times.
Tissue culture and VLP-budding assays
Human embryonic kidney 293T cells were grown in 2 ml Dulbecco's minimum essential medium (6-well plates) and were transfected with expression plasmids as indicated in the figure legends using LipofectAMINETM 2000 (Invitrogen; Garrus et al., 2001). VLP-budding assays were performed essentially as described previously (Garrus et al., 2001). In brief, plasmid DNA encoding GagGFP and GagHrs fusion proteins was transfected into 293T cells. Supernatants and cytoplasmic lysates were harvested 2428 h later and analyzed by Western blotting (see below).
Functional siRNA knockout and rescue of Tsg101 expression were performed as described previously (Elbashir et al., 2001; Garrus et al., 2001). Synthetic 21-nt siRNA duplexes were designed to target Tsg101 at coding nucleotides 413433. siRNA sequences: sense, CCU CCA GUC UUC UCU CGU CdTdT; antisense, 5' GAC GAG AGA AGA CUG GAG GdTdT. 293T cells were cotransfected twice at 24-h intervals. The first transfection was with 50 nM siRNA duplexes and either 2 µg pIRES2-EGFP or 2 µg wild-type or mutant RNAi-resistant pIRES-Tsg*Flag expression vector (Tsg101*). The second transfection was performed with the same 50-nM siRNA duplexes and 1 µg Gagp6Hrs
N expression vector. VLP-containing supernatants and cytoplasmic lysates were harvested after an additional 2430 h and analyzed by Western blotting (see below).
Western blots
VLPs from 1.2 ml of supernatants from transfected cells were pelleted through a 20% sucrose cushion in a microcentrifuge for 90 min at 13,000 rpm (4°C) and resuspended in 2530 µl of 1x SDS gel loading buffer. 58-µl samples were separated by SDS-PAGE, transferred, blocked, blotted with antisera, and protein bands were detected by ECL (Pierce Chemical Co.). Transfected 293T cells (one well from a 6-well plate) were harvested directly into 3035 µl RIPA buffer (10 mM TrisCl, pH 7.0, 150 mM NaCl, 1% NP-40, and 0.1% SDS) and incubated on ice for 4 min. Samples were clarified by microcentrifugation for 4 min at 13,000 rpm (4°C), and resuspended in an equal volume of 2x SDS gel loading buffer. 58-µl aliquots were resolved by SDS-PAGE and blotted for ECL. To detect Gag proteins, rabbit anti-HIV CA antibody (1:2,000; from Hans-Georg Krausslich, Heidelberg, Germany) was mixed with rabbit anti-HIV MA (1:25,000; from Didier Trono, Geneva, Switzerland). Murine monoclonal anti-Tsg1014A10 (1:1,000; GeneTex, Inc.) was used to detect Tsg101.
Electron microscopy
VLPs from 80 ml pooled culture supernatant were pelleted through 20% sucrose cushions and prepared for thin-section EM as described previously (von Schwedler et al., 1998). Cells were prepared for thin-section EM as described previously (Garrus et al., 2001). Samples were imaged on a transmission electron microscope (Tecnai-12; Philips).
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Acknowledgments |
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This work was supported by an National Institutes of Health grant to W.I. Sundquist.
Submitted: 24 February 2003
Accepted: 4 June 2003
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References |
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