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Journal of Virology, July 2005, p. 8933-8941, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8933-8941.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

AIP1/Alix Is a Binding Partner of Sendai Virus C Protein and Facilitates Virus Budding

Takemasa Sakaguchi,1* Atsushi Kato,2 Fumihiro Sugahara,1 Yukie Shimazu,1 Makoto Inoue,3 Katsuhiro Kiyotani,1 Yoshiyuki Nagai,4 and Tetsuya Yoshida1

Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551,1 Department of Virology 3, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo 208-0011,2 DNAVEC Corporation, Tsukuba, Ibaraki 305-0856,3 Toyama Institute of Health, Imizu-gun, Toyama 939-0363, Japan4

Received 26 January 2005/ Accepted 24 March 2005


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ABSTRACT
 
The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.


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INTRODUCTION
 
Sendai virus (SeV), the prototype of the paramyxoviruses, produces enveloped progeny virions by budding from the plasma membrane. SeV has six genes, N, P, M, F, HN, and L, on its nonsegmented single-stranded RNA genome in a tandem arrangement from which six proteins are individually synthesized. Among these viral proteins, the M protein is the central organizer in paramyxovirus budding and virus morphogenesis. The M protein self-associates and interacts with membranes, forming patches at the inner surface of the plasma membrane (2, 39). The M protein also interacts with the glycoproteins F and HN and viral nucleocapsids (1, 49, 50). In vitro mutagenesis of the M protein has demonstrated its involvement in virus morphogenesis and budding (14, 26, 34). Furthermore, the M protein forms vesicles and self-releases from cells when singly expressed from cDNA (33, 40, 42). The SeV glycoproteins F and HN are also important for budding, since both modulate the release and integrity of virus-like particles (VLPs) formed by coexpression of multiple viral proteins (40). The F protein has a self-release activity when expressed alone (42), and the cytoplasmic tails of the F and HN proteins are essential for regulating morphogenesis and budding (7, 41).

In addition to the six proteins, SeV encodes accessory proteins, namely, the C proteins and the V protein (reviewed in references 21 and 27). It is considered that the C proteins are not involved in virus assembly, since they are essentially nonstructural components that are abundantly expressed in infected cells but are also present in trace amounts in mature virions. However, evidence is emerging that the C protein may be involved in the virus budding process.

The SeV C proteins are expressed from the P gene as a nested set of four carboxyl-coterminal proteins (C', C, Y1, and Y2). They are nonessential, since a knockout virus lacking the C proteins was successfully generated, although the virus was unable to grow efficiently in cultured cells or in mice (20; reviewed in reference 27). The C proteins have at least three capabilities: disturbance of interferon signaling, inhibition of beta interferon induction, and suppression of viral RNA synthesis (3, 5, 6, 10, 13, 30). In addition, the C-protein knockout [4C(–)] virus produced largely noninfectious progeny virions with a highly anomalous morphology (11). Excessive accumulation of viral proteins and genomic RNAs was also observed in 4C(–) virus-infected cells (11). These results suggest that the late step of budding is abrogated in the absence of the C protein. We recently reported that simultaneous expression of the M, N, F, and HN proteins in cells resulted in the formation of VLPs similar to authentic virus particles in their density and morphology. We further showed that an additional expression of the C protein with these four proteins resulted in an increase in VLP production, indicating that the C protein has a budding-enhancing activity (40). The C protein is thus thought to facilitate the late step of virus budding. However, the precise role of the C protein in virus maturation is still an enigma.

In the present study, we identified AIP1/Alix as an interacting partner of the C protein, and their interaction was found to be involved in efficient budding of VLPs. We also showed that AIP1/Alix is necessary for efficient SeV budding.


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MATERIALS AND METHODS
 
Yeast two-hybrid screening. PCR-amplified DNAs encoding full-length SeV C (204 amino acids), Y2 (residues 30 to 204), and Y3 (residues 99 to 204) proteins were subcloned in frame with the GAL4 DNA-binding domain in the vector pDBLeu (Invitrogen) to generate pDBLeu-C, pDBLeu-Y2, and pDBLeu-Y3, respectively. A HeLa cell cDNA library (Invitrogen) constructed in the vector pPC86 under the GAL4 activation domain was used for screening protein-protein interactions. Recombinant pDBLeu-C (10 µg) and pPC86-HeLa cDNAs (10 µg) were introduced into the yeast strain MaV203 according to the manufacturer's protocol. Aliquots of the cells were plated on solid synthetic defined (SD) medium lacking Trp and Leu, and the library size was estimated to be 1.3 x 106 colonies. For selection of double transformants, cells were plated onto SD plates lacking Trp, Leu, and His in the presence of the His biosynthesis inhibitor 3-amino-1,2,4-triazole (3AT) at 25 mM. Colonies grown on the selection plates were selected, and the plasmids were extracted and sequenced. To confirm the interaction, the plasmid pDBLeu-Y2 or pDBLeu-Y3 and the pPC86-based vector encoding a candidate protein were introduced into MaC203 cells. To measure the strength of protein-protein interactions, cells were grown on SD plates lacking Trp, Leu, and His in the presence of various amounts of 3AT (0 to 100 mM).

Plasmid preparation. The full-length cDNA clone of AIP1/Alix (DDBJ/EMBL/GenBank accession numbers BC020066 and AF151793) was obtained from Open Biosystems and subcloned into either the pCAGGS vector under the chicken ß-actin promoter (28) or the pKS336 vector under the human elongation factor promoter (31) with simultaneous addition of a hemagglutinin (HA) tag at the N terminus or C terminus. The N-terminally HA-tagged deletion mutants AIP11-702 and AIP1358-868 (see Fig. 3A) were subcloned into pCAGGS. The recombinant clones were confirmed by sequencing.



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  		FIG. 3. Interaction of the C protein with the N-terminal region of AIP1/Alix. (A) Schematic view of AIP1/Alix. The deletion mutants AIP11-702 and AIP1358-868 are also shown. The positions of the Bro-like domain and the cysteine-rich domain were described previously by Shibata et al. (37). (B) Expression plasmids for HA-AIP1, HA-AIP11-702, and HA-AIP1358-868 or an empty vector (vec) were introduced into 293T cells together with the C-protein expression plasmid. Proteins were metabolically labeled with [35S]cysteine and [35S]methionine and solubilized in a mild detergent. The sample was divided into two parts that were precipitated with either an anti-HA antibody ({alpha}HA) or an anti-C-protein antibody ({alpha}C) and then analyzed by SDS-PAGE.

The pCAGGS-M, pCAGGS-N, pCAGGS-F, pCAGGS-HN, and pCAGGS-C plasmids for expression of SeV proteins were described previously (33, 40). The cDNA of the C protein was subcloned into pKS336 to generate pKS336-C (15), and in vitro mutagenesis was performed on the plasmid by using a QuickChange XL mutagenesis kit (Stratagene). The expression plasmids for green fluorescent protein (GFP)-conjugated Vps4A and Vps4A-E228Q were described previously (8).

Cells and viruses. 293T cells (human renal epithelial cells expressing the simian virus 40 large T antigen) were propagated in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal calf serum. LLC-MK2 and CV1 cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Wild-type SeV derived from a cDNA of the Z strain (16) and its 4C(–) mutant virus, in which all four C proteins were knocked out (20), were propagated in embryonated chicken eggs, and infectivity was measured using an immunofluorescent infectious focus assay (19) and expressed as cell infectious units (CIU)/milliliter.

Coimmunoprecipitation and Western blotting. Subconfluent 293T cells were transfected with plasmids by using TransFectin (Bio-Rad Laboratories), and after 24 h, the cells were solubilized with 0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and a "complete" protease inhibitor cocktail (Roche Diagnostics). Proteins were immunoprecipitated with either an anti-SeV serum plus an anti-C-protein serum or an anti-HA antibody (262K; Cell Signaling Technology). The immunoprecipitated proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using a 10% gel, and the protein bands were visualized and quantified using a BAS2000 Bio-imaging Analyzer (Fuji Film) as described previously (32). Western blotting was performed as described previously (33) using the anti-HA antibody.

Immunofluorescent staining. SeV-infected and pKS336-HA-AIP1-transfected CV1 cells were fixed with 0.5% methanol-free formaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min at 18 h after infection. The cells were then treated with 1 M glycine in PBS and 0.1% Triton X-100 in PBS and incubated with an anti-HA monoclonal antibody and either an anti-C rabbit serum or an anti-P rabbit serum. The cells were subsequently incubated with an Alexa 488-conjugated anti-mouse immunoglobulin antibody and an Alexa 546-conjugated anti-rabbit immunoglobulin G antibody (Molecular Probes) and observed under a confocal microscope (Axiovert 100 M; Carl Zeiss).

VLP formation in the presence of mutant C proteins. VLP formation was performed as described previously (40). Briefly, subconfluent 293T cells in 60-mm dishes were transfected with a mixture of pCAGGS-M, pCAGGS-F, pCAGGS-N, and pCAGGS-HN together with pKS336-C for expression of wild-type (WT) C protein or one of its mutants. After 24 h, the medium of the transfected 293T cells was replaced with 1.5 ml of Dulbecco's modified Eagle's minimal essential medium containing 1/10 of the normal amounts of cysteine and methionine and 2.5 MBq/ml of a mixture of [35S]cysteine and [35S]methionine ([35S]Pro-mix; Amersham Biosciences).

After 48 h, the medium and cells were collected separately. VLPs in the medium were concentrated by ultracentrifugation with an RPS40T rotor (Hitachi) at 35,000 rpm for 1 h, and the viral proteins in the pellet were solubilized with a radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 150 mM NaCl) containing a protease inhibitor cocktail. Cells were solubilized in the radioimmunoprecipitation assay buffer and then centrifuged at 15,000 rpm for 20 min to remove the nuclei and cell debris. Viral proteins in the fractions were precipitated with an anti-SeV serum and an anti-C serum and analyzed by SDS-PAGE. The VLP budding rates were calculated as the ratio of the M protein detected in the medium to the total M protein in both the medium and the cells (40).

SeV budding in overexpression of AIP1/Alix. Subconfluent 293T cells in 60-mm dishes were transfected with pCAGGS-HA-AIP1, pCAGGS-HA-AIP11-702, or pCAGGS-HA-AIP1358-858. After 16 h, the cells were further infected with SeV or 4C(–) virus at a multiplicity of infection of 10. The cells were then metabolically labeled with a mixture of [35S]cysteine and [35S]methionine from 24 h through 48 h after transfection, and the viral proteins in the medium and the cells were analyzed as described above.

Depletion of AIP1/Alix by small interfering RNA. Synthetic oligonucleotides were inserted between the human U6 promoter and the terminator sequence of the pBAsi-hU6 vector (Takara) to generate a stem-loop type of small interfering RNA in transfected cells. pBAsi-AIP1, which targeted nucleotides 1313-GAACTGCCTGAATTACTGC-1331 corresponding to the AIP1/Alix coding frame, was constructed, and pBAsi-NC, which targeted a randomized nucleotide sequence, was constructed as a negative control. To assess the depletion of AIP1/Alix, 293T cells were transfected with the expression plasmid for HA-AIP1 (pKS336-HA-AIP1) and either pBAsi-AIP1 or pBAsi-NC. After 24 h, the cells were metabolically labeled with [35S]cysteine and [35S]methionine for 30 min and then processed by immunoprecipitation with an anti-HA antibody and SDS-PAGE. The HA-AIP1 bands were quantified using an image analyzer. To investigate the effect of pBAsi-AIP1 on SeV budding, 293T cells in 60-mm dishes were transfected with 2 µg of the pBAsi vector at 2 days prior to infection and again on the day of infection. The cells were then infected with SeV, and the viral proteins were metabolically labeled from 24 h through 48 h after infection and analyzed as described above.

SeV budding in coexpression of Vps4A. SeV nucleocapsids were purified as described previously (5). Briefly, purified virions were solubilized in 1% Triton X-100 and 1 M KCl in PBS and concentrated onto 68% (wt/vol) sucrose-D2O through 50% glycerol, 30 mM NaCl, and 10 mM HEPES (pH 7.4) by ultracentrifugation with an SW55 rotor at 43,000 rpm for 90 min. The collected nucleocapsids, containing the N, P, and L proteins, could initiate virus replication when transfected into cells. The expression plasmid for Vps4A-WT or Vps4A-E228Q (1 µg) was introduced into subconfluent CV1 cells in 35-mm dishes together with the purified SeV nucleocapsids (5 µg) using 4 µl of Lipofectamine (Invitrogen) according to the manufacturer's protocol, and the medium was harvested after 24 h. Infectivity in the medium was measured after trypsin treatment as described previously (33). Western blotting of the cell lysates was performed as described previously (33) using an anti-GFP antibody (sc-8334; Santa Cruz Biotechnology), an anti-actin antibody (MAB1501; Chemicon), and an anti-SeV rabbit serum.


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RESULTS
 
AIP1/Alix interacts with the C protein. We searched for an interacting partner of the C protein by using a yeast two-hybrid system and obtained 120 positive clones from a library of 1.3 x 106 clones. Five of the positive clones were revealed to possess the cDNA for ALG-2-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix).

When Y2 (a natural variant corresponding to residues 30 to 204 of the C protein) or Y3 (residues 99 to 204) (Fig. 1) fused with the GAL4 DNA-binding domain was used as the bait and AIP1 lacking 9 amino acids at the N terminus, probably caused during the cDNA cloning procedure, fused with the GAL4 DNA activation domain was used as the prey, yeast could grow despite increased amounts of 3AT to as much as 100 mM (Fig. 1). These results indicate that Y3 as well as Y2 can interact with AIP1 in the nucleus of yeast cells and that the carboxyl half of the C protein (106 amino acids) is important for the interaction.



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  		FIG. 1. Interaction of the C protein with AIP1/Alix in yeast cells. (A) Schematic view of the C proteins (C', C, Y1, and Y2) and a deletion mutant (Y3). (B) Growth of yeast cells possessing AIP1 as the prey and Y2 or Y3 as the bait in the presence of 3AT (0 to 100 mM).

N-terminally or C-terminally HA-tagged AIP1 was expressed in mammalian cells together with the SeV proteins M, N, F, HN, and C, which are minimally required for generating VLPs that resemble authentic virions with high efficiency of budding (40). Metabolic labeling and immunoprecipitation revealed that an anti-HA antibody precipitated not only the HA-tagged AIP1 but also the C protein (Fig. 2A). In this experiment, the anti-HA antibody precipitated N-terminally-tagged AIP1 to a greater extent than the C-terminally-tagged AIP1, and the amounts of the precipitated C protein corresponded to those of AIP1 (Fig. 2A). Conversely, the C protein pulled down AIP1 (Fig. 2A and B), and the C protein in virus-infected cells also pulled down AIP1 (Fig. 2B), suggesting that the C protein and AIP1 interact not only in yeast cells but also in mammalian cells. In addition, immunofluorescent staining demonstrated that AIP1 was colocalized with the C protein in the cytosol and cellular membrane of SeV-infected and AIP1-transfected cells (Fig. 2C), while AIP1 was colocalized with the P protein only partly (Fig. 2C).



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  		FIG. 2. Interaction of the C protein with AIP1/Alix in mammalian cells. (A) N-terminally or C-terminally HA-tagged AIP1 (HA-AIP1 and AIP1-HA, respectively) was expressed in 293T cells together with SeV proteins. Proteins were labeled with [35S]cysteine and [35S]methionine, solubilized in a mild detergent, precipitated with an anti-HA antibody ({alpha}HA) or a mixture of anti-SeV and anti-C protein antibodies ({alpha}SeV+{alpha}C), and analyzed by SDS-PAGE. (B) HA-AIP1 and the C protein were coexpressed in 293T cells (upper panel). Alternatively, HA-AIP1 was expressed in SeV-infected cells (lower panel). Proteins were analyzed as described above (A). (C) HA-AIP1 was expressed in SeV-infected cells. The cells were then stained with an anti-C-protein antibody (red, Alexa 546) and an anti-HA antibody (green, Alexa 488) and observed by confocal microscopy. An anti-P antibody (red, Alexa 546) was used as a control.

To investigate which region of AIP1 (868 amino acids) is required for the binding to the C protein, two deletion mutants were tested. A polypeptide of AIP1 at positions 1 to 702, containing the Bro-like domain, was coprecipitated with the C protein and required for the interaction (Fig. 3). In contrast, a polypeptide of the carboxyl terminus of AIP1 at positions 358 to 868, containing a proline-rich domain and the ALG-2-binding site, was not precipitated with the C protein, indicating that the polypeptide at positions 1 to 357 may be critical for the interaction with the C protein.

Acceleration of VLP release by the C protein is correlated with the interaction with AIP1. The regions of the C protein required for interaction with AIP1 were investigated. The naturally occurring N-terminal variants C', C, Y1, and Y2 all precipitated AIP1 (Fig. 4A and B). In contrast, a mutant C protein with a 5-amino-acid deletion at the carboxyl terminus (d199) pulled down AIP1 only slightly, and mutant C proteins with a deletion of 10 amino acids or more at the carboxyl terminus (d194, d189, d184, d181, and d126 [mutants shown in Fig. 4A]) did not pull down AIP1 at all (Fig. 4B). Together with the results of the yeast two-hybrid experiment (Fig. 1), it is suggested that the C terminus of the C protein is important for the interaction with AIP1.



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  		FIG. 4. Correlation between binding of the C protein with AIP1 and the ability to accelerate VLP release. (A) Schematic view of the mutant C proteins. The interaction with AIP1 and acceleration of VLP release of the mutant C proteins are shown in the figure as +, ±, and –. (B) Coprecipitation of AIP1 with the C protein. HA-AIP1 and a mutant C protein were coexpressed in 293T cells. Proteins were labeled, precipitated with an anti-C-protein antibody ({alpha}C), and analyzed by SDS-PAGE. vec, empty vector. Proteins were also precipitated with an anti-HA antibody ({alpha}HA) to mark the migration position of HA-AIP1. (C, D) Acceleration of VLP release by the C protein. VLPs were generated by simultaneous expression of the F, HN, M, and N proteins together with a mutant C protein as indicated. After metabolic labeling with [35S]cysteine and [35S]methionine, VLPs in the medium were concentrated by ultracentrifugation and processed for immunoprecipitation with anti-SeV and anti-C antibodies. Cells were solubilized and also processed for immunoprecipitation. The M-protein bands in both the medium and the cells were quantified using an image analyzer, and the VLP budding rates were calculated. The ratios of the released M-protein fractions are shown in the figure (C) and in the graph (D) with coexpression of the empty vector (vec) as 1.0. Proteins from 1/30 of the cell lysates and 1/3 of the medium were run in the different lanes (C).

The C protein has been shown to accelerate VLP formation by two- to threefold in a VLP generation system (40). The ability of the C mutants to accelerate VLP release was therefore investigated. As shown in Fig. 4C, viral proteins including the C protein were released into the medium. The fraction of the M protein released into the medium increased by almost threefold in the presence of the wild-type C protein (Fig. 4C). In contrast, the fractions of the M protein released in the presence of the C-terminal deletion mutants d199, d194, d189, and d184 were similar to those released without the C protein (Fig. 4C). The M protein was used because it is a central player in virus budding and considered to reflect virus budding (40). Release rates based on the HN proteins had a tendency similar to that based on the M protein, whereas release rates based on the F and N proteins were partly enhanced in the presence of the deletion mutants of the C protein.

In this case, the C protein was detected in the medium fraction and appeared to be more abundant than that in the cells. Some C proteins have been reported to be incorporated into virions (48), and the C protein bands from the medium may be augmented by the fact that there was a 10-fold difference in the loading amounts; proteins from 1/30 of the cell lysates and 1/3 of the medium were loaded in the different lanes.

On the other hand, in the case of the naturally occurring N-terminal variants C', C, Y1, and Y2, the fraction of the M protein released was greater than that released without the C protein, although there were differences in the extent of the release enhancement (Fig. 4D). In these variants, the C protein showed the greatest capacity to support VLP budding, suggesting that it provides an advantage for virus budding. This may be one reason why the C protein is the most abundant protein in virus-infected cells among the naturally occurring N-terminal variants. Figure 4A, which summarizes the results of Fig. 4B, C, and D, shows that the ability of the C proteins to accelerate VLP release corresponds to their coprecipitation with AIP1, suggesting the importance of the interaction between the C protein and AIP1 for the budding process.

Acceleration of SeV budding by overexpression of AIP1/Alix. We next investigated the effect of AIP1/Alix on SeV budding by overexpressing AIP1/Alix in SeV-infected cells. 293T cells were transfected with an AIP1 expression plasmid and subsequently infected with SeV. Analysis of the fractions of the M protein released into the culture medium revealed that AIP1 increased the amount of released SeV by approximately 3.7-fold (Fig. 5A). The N-terminal polypeptide at positions 1 to 702 facilitated SeV budding, whereas the C-terminal peptide at positions 358 to 868 did not (Fig. 5B). This facilitation correlated to the capacity of AIP1 to bind to the C protein. Certain amounts of AIP1 and the peptide at positions 1 to 702 were also detected in the medium fraction by immunoblotting (Fig. 5B). This facilitation of virus release was not observed when the 4C(–) virus was infected instead of the wild-type virus (Fig. 5C), suggesting that the acceleration of virus release was dependent on the presence of the C protein.



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  		FIG. 5. Acceleration of SeV release by overexpression of AIP1/Alix. (A) HA-AIP1 was expressed in SeV-infected 293T cells. Cells were labeled with [35S]cysteine and [35S]methionine from 24 h through 48 h posttransfection, and the virus particles in the medium and viral proteins in the cells were separately harvested as described in the legend for Fig. 4. Proteins were precipitated with anti-SeV plus anti-C antibodies and analyzed after SDS-PAGE using an image analyzer. The virus budding rates were calculated. The ratios of the released M fractions are shown with coexpression of the empty vector (vec) as 1.0. (B) HA-AIP11-702 and HA-AIP1358-868 were examined for their abilities to accelerate SeV release as described above (A). Proteins were precipitated with an anti-HA antibody ({alpha}HA) from aliquots of the cell lysates. Aliquots of the pellets from the medium fractions were blotted onto a membrane and probed with an anti-HA antibody to detect HA-AIP mutant proteins in the fraction (WB: {alpha}HA). An asterisk indicates a truncated form of AIP1. (C) A similar experiment was performed using a C-protein knockout virus [4C(–) virus] instead of wild-type SeV. (D) HA-AIP1 was expressed in SeV-infected 293T cells, and the cells were labeled with [35S]cysteine and [35S]methionine from 24 h through 48 h posttransfection. The medium was then separated by equilibrium ultracentrifugation in a continuous 10 to 50% sucrose gradient. The proteins in each fraction were analyzed by immunoprecipitation with a mixture of anti-SeV, anti-C-protein, and anti-HA antibodies, followed by SDS-PAGE. The density of each fraction is shown. The identity of HA-AIP1 was confirmed by immunoprecipitation with the anti-HA antibody alone (data not shown).

To characterize the virus released from the cells, we fractionated it by equilibrium ultracentrifugation in a continuous 10 to 50% (wt/wt) sucrose-PBS gradient as described previously (40) and found that large amounts of nucleocapsid-free empty virions (fractions 9 and 10) were released in addition to mature virus particles (fractions 13 to 15) from AIP1-overexpressing SeV-infected cells (Fig. 5D). This was not previously seen in wild-type SeV-infected cells (40), but a similar enhancement of empty virion release was observed when the M protein of vesicular stomatitis virus was overexpressed in SeV-infected cells (33). The current result suggests that overexpression of AIP1 promotes the release of virions composed of the viral glycoproteins and the matrix proteins F, HN, and M but does not promote nucleocapsid incorporation into these virions. Furthermore, a large amount of AIP1 was found in the low-density vesicles near the top fraction of the gradient, and the C protein was also detected near this fraction (Fig. 5D).

Inhibition of SeV budding by AIP1/Alix depletion. To investigate the involvement of AIP1 in SeV budding, we depleted the intracellular AIP1 using a plasmid that synthesized a small interfering RNA targeting AIP1 mRNA (pBAsi-AIP1). The plasmid inhibited the synthesis of cotransfected tagged AIP1 to almost 1% (Fig. 6A), indicating that new synthesis of AIP1 was abrogated by the plasmid. 293T cells were transfected with pBAsi-AIP1 twice with an interval of 2 days in order to deplete intrinsic AIP1 and then infected with SeV. At 24 h after the infection, viral proteins were metabolically labeled for a further 24 h, and the proteins released into the culture medium were analyzed. SeV release from the infected cells was about 36% of that from control cells (Fig. 6B and C). These findings demonstrate that although AIP1 is not essential, it is critical for efficient SeV budding.



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  		FIG. 6. Suppression of SeV release by depletion of AIP1/Alix with small interfering RNA. (A) 293T cells were transfected with an expression plasmid for HA-AIP1 (pKS336-HA-AIP1) and either pBAsi-AIP1 or pBAsi-NC. After 24 h, the cells were metabolically labeled with [35S]cysteine and [35S]methionine for 30 min and processed by immunoprecipitation with an anti-HA antibody and SDS-PAGE. The HA-AIP1 bands were quantified using an image analyzer. (B) 293T cells were transfected with pBAsi-AIP1 or pBAsi-NC. After 2 days, the cells were transfected with the same plasmids again and then infected with SeV at an input multiplicity of infection of 10. After 24 h, the cells were metabolically labeled with [35S]cysteine and [35S]methionine for a further 24 h. The medium and cells were harvested separately and analyzed as described in the legend for Fig. 4. The ratios of the released M-protein fractions are shown with coexpression of pBAsi-NC as 1.0. The graph shows the ratios of the M-protein fractions released into the medium from three independent experiments. The error bars indicate the standard deviations.

Effect of Vps4A on SeV budding. AIP1/Alix has been shown to interact with the Gag proteins of retroviruses, and these interactions are presumed to result in the recruitment of endosome-associated complexes required for transport (ESCRT) to virus budding (22, 38, 47). To investigate whether ESCRT is involved in SeV budding, a dominant-negative form of the Vps4A AAA-type ATPase, an essential component of ESCRT, was employed. Transfection of the dominant-negative form, Vps4A-E228G, and infectious purified SeV nucleocapsids into CV1 cells yielded a significantly lower titer of infectious virus in the medium after 24 h (Fig. 7A) than that of wild-type Vps4A and a mock plasmid. Expression of the Vps4 plasmids was confirmed by Western blotting (Fig. 7B). A similar experiment was performed in another efficient SeV producer, LLC-MK2 cells, and the same results were obtained (data not shown). In this experiment, nucleocapsid transfection was used as a surrogate for virus infection for the following reason. Due to the low DNA transfection efficiency in CV1 cells, there were a number of SeV-replicating, but not plasmid-transfected, cells when the cells were infected with SeV. In contrast, a considerable overlap between virus-replicating and plasmid-transfected cells was observed when nucleocapsids and plasmid DNA were introduced by transfection (data not shown). These results suggest that ESCRT is involved in SeV budding.



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  		FIG. 7. Suppression of SeV release by a Vps4A mutant. A plasmid expressing GFP-conjugated wild-type Vps4A (Vps4A-WT) or a dominant-negative form of Vps4A (Vps4A-E228Q) was introduced into CV1 cells together with purified infectious SeV nucleocapsids, and the medium was harvested after 24 h. (A) The graph indicates the infectivities in the medium from three independent dishes. The error bars indicate the standard deviations, and the asterisks indicate a significant difference (*P < 0.05, **P < 0.01; Student's t test). (B) Western blotting of the cell lysates was preformed using an anti-GFP ({alpha}GFP) antibody to detect GFP-Vps4A fusion proteins (689 amino acids), an anti-actin antibody ({alpha}actin), or an anti-SeV ({alpha}SeV) antiserum. Migrating positions of protein mass markers (in kilodaltons) are shown.

We performed a VLP budding assay by expressing N, M, F, HN, and C proteins in 293T cells together with a Vps4A protein. Vps4A-WT caused strong suppression of VLP release, and the VLP release was rather restored by Vps4A-E228G (data not shown). This observation suggests a relation of Vps4A with VLP budding, but this must be investigated further.


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DISCUSSION
 
In the present study, we have shown that the SeV C protein interacts with AIP1/Alix and that this interaction is involved in VLP budding, since mutant C proteins unable to bind to AIP1 failed to accelerate VLP budding. We further investigated the involvement of AIP1 in SeV budding. First, overexpression of AIP1 facilitated SeV budding, and this was not observed for either 4C(–) virus or the AIP1 mutant unable to bind the C protein. Second, depletion of AIP1 by small interfering RNA suppressed SeV budding. These results suggest that AIP1 functions in SeV budding through interaction with the C protein. Although the C proteins are not efficiently incorporated into virus particles (48), they colocalize with the M protein and the F and HN glycoproteins in infected cells (11).

AIP1/Alix has been shown to interact with human immunodeficiency virus type 1 p6 and equine infectious anemia virus p9 (22, 38, 47). These interactions are presumed to result in the recruitment of ESCRT, the protein network involved in endosome transport and multivesicular body (MVB) formation (18), to virus budding and subsequently facilitate these viruses to pinch off from cells at the late step of infection. AIP1/Alix has been shown to be a key component that connects the proteins of ESCRT-1 with those of ESCRT-3 (17, 22, 29, 47). MVB formation involves membrane restructuring accompanying membrane budding into the late endosome. Since the topology of MVB membranes is equivalent to virus budding, host factors involved in MVB formation are also likely to be involved in virus budding (12, 25). It therefore seems reasonable to hypothesize that the C protein has a similar function for SeV budding. In order to test this hypothesis, we investigated whether SeV required ESCRT for its budding. As shown in Fig. 7, a dominant-negative form of Vps4A, an AAA-type ATPase essential for ESCRT function (8), inhibited SeV budding. This result suggests that ESCRT is also necessary for efficient budding of the paramyxovirus SeV. Inhibition of virus budding by the dominant-negative form of Vps4A has been described previously in another paramyxovirus, simian virus 5 (36).

The results of the present study further suggest that the C protein contains the L domain. In that case, the L domain is presumed to reside in the C-terminal half (106 amino acids) of the C protein, although this region does not contain any of the known amino acid sequence motifs required for interaction with AIP1/Alix, namely, LYPXXXL, LYPXL, and LXXLF (38). From the results of this study using deletion mutants, we expected that the C terminus of the C protein contained amino acid residues important for the interaction with AIP1. However, extensive alanine scanning of this region failed to identify such residues (data not shown). The C-protein motif essential for its interaction with AIP1 in other regions is now under investigation.

AIP1/Alix was originally identified as an interacting partner of apoptosis-linked gene 2 (ALG-2), which is involved in apoptosis of neuronal cells (45) and has also been shown to be involved in apoptosis (4, 24, 44, 46). However, budding acceleration by AIP1 may not be directly related to apoptosis, since there was no evidence that AIP1 overexpression induced apoptosis in 293T cells (data not shown) and acceleration of SeV budding was induced by overexpression of the polypeptide at positions 1 to 702 lacking the ALG-2-binding site (37, 44), which is essential for inducing apoptosis (44). On the other hand, AIP1/Alix is a component of ESCRT, as stated above. Furthermore, there is evidence that AIP1/Alix is actively involved in membrane restructuring. AIP1/Alix is incorporated into an MVB-resembling membrane structure generated in vitro in the presence of an appropriate membrane pH gradient and a specific phospholipid, lysobisphosphatidic acid (23), and has also been detected in small membrane structures, designated exosomes, that are liberated from cells (9, 43). In the case of overexpression of AIP1 in SeV-infected cells, abundant low-density liposomes containing AIP1 and the C protein were observed near the top fraction by equilibrium ultracentrifugation in a sucrose gradient (Fig. 5D). AIP1 may be directly involved in membrane vesicle release from cells, and the fact that AIP1/Alix interacts with actin may be related to this effect (35). The acceleration of virus budding by the interaction between the C protein and AIP1/Alix could therefore be caused by the direct effect of AIP1 to generate vesicles as well as by recruiting ESCRT to virus budding.

AIP1 was found to coprecipitate with the C proteins of human parainfluenza virus 1 and human parainfluenza virus 3 but not with the C protein of measles virus (data not shown). Involvement of AIP1 in virus budding may be true for other members of the genus Respirovirus but is not true for the members of the genus Morbillivirus in the subfamily Paramyxovirinae.


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ACKNOWLEDGMENTS
 
We thank E. Suzaki (2nd Department of Anatomy, Hiroshima University) for her kind help with the confocal microscopy. We also thank J. E. Garrus and W. I. Sundquist (University of Utah, UT) for providing the Vps4A expression vectors and K. Sakai and S. Morikawa (National Institute of Infectious Diseases, Japan) for providing the pKS336 vector.

We also thank the staff of the 1st Department of Anatomy, the Research Center for Molecular Medicine, and the Analysis Center of Life Science, Hiroshima University, for the use of their facilities. This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: 81-82-257-5157. Fax: 81-82-257-5159. E-mail: tsaka{at}hiroshima-u.ac.jp. Back


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Journal of Virology, July 2005, p. 8933-8941, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.8933-8941.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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