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1

2

3

4

YLDL Sequence within Sendai Virus M protein is Critical for5

Budding of Virus-Like Particles and Interacts with Alix/AIP16

Independently of C protein7

8

9

Takashi IRIE*, Yukie Shimazu, Tetsuya Yoshida, and Takemasa Sakaguchi10

11

12

Department of Virology, Graduate School of Biomedical Sciences, Hiroshima 13

University, Hiroshima 734-8551, Japan14

15

16

17

*Corresponding author: Department of Virology, Graduate School of Biomedical 18

Sciences, Hiroshima University, 1-2-3 Kasumi, Munami-ku, Hiroshima 734-8551,19

Japan20

Phone: 81-82-257-5157; Fax: 81-82-257-5159; E-mail: tirie@hiroshima-u.ac.jp21

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Abstract1

For many enveloped viruses, cellular multivesicular body (MVB) sorting 2

machinery has been reported to be utilized for efficient viral budding. Matrix and 3

Gag proteins have been shown to contain one or two L-domain motifs (PPxY, 4

PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular 5

proteins involved in multivesicular body sorting, which are recruited to the viral 6

budding site. However, for many enveloped viruses, L-domain motifs have not yet 7

been identified, and the involvement of MVB sorting machinery in viral budding is 8

still unknown. Here we show that both Sendai virus (SeV) matrix protein M and 9

accessory protein C contribute to virus budding by physically interacting with 10

Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and 11

its specific interaction with the N-terminus of Alix/AIP1 (1-211) was important for 12

the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP 13

budding was inhibited by the overexpression of some deletion mutants of Alix/AIP1 14

and depletion of endogenous Alix/AIP1 using specific siRNAs. The YLDL 15

sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, 16

and even YPxL. C protein was also able to physically interact with the N-terminus17

of Alix/AIP1 (212-357) and enhanced M-VLP budding independently of 18

M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. 19

Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may 20

enhance the efficiency of the utilization of cellular MVB sorting machinery for 21

efficient SeV budding.22

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Introduction1

Enveloped viruses bud from cellular membranes to acquire lipid-containing2

envelopes, and require a membrane fission event to release virions from host cells at 3

the final step of their lifecycle. Viral matrix proteins such as Gag for retroviruses, 4

VP40 for filoviruses, and M for rhabdoviruses have been shown to be able to bud 5

from the cell surface by themselves in the form of lipid-enveloped, virus-like6

particles (VLPs), suggesting that these proteins play important roles in the 7

late-budding step (23, 25, 28, 40, 49). Subsequent investigations have shown that 8

these proteins possess late-budding (L) domains, which are critical for efficient 9

budding. To date, three consensus sequences of L-domains have been identified 10

within these matrix proteins (5). The majority of retroviruses possess PPxY- and/or 11

PT/SAP-type L-domain motifs except for equine infectious anemia virus (EIAV), 12

which possesses a YPxL-type L-domain motif (11). Rhabdoviruses such as 13

vesicular stomatitis virus (VSV) and rabies virus (RV) possess a PPPY motif within 14

M proteins, and Ebola virus possesses overlapping L-domain motifs (7-PTAP-10 and 15

10-PPEY-13) within VP40 (15, 22, 24, 34). More recently, another potential 16

L-domain motif, LxxL, has been identified within HIV-1 p6 and EIAV p9, here 17

overlapping the YPxL motif (45).18

The cellular interacting partners of these L-domain motifs have also been 19

identified. The PPxY motifs of retroviruses, rhabdoviruses, and filoviruses, and the 20

PT/SAP motifs of HIV-1 and Ebola virus have been shown to interact with 21

Nedd4-like E3 ubiquitin ligases via their WW domains and tumor susceptibility gene 22

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101 (Tsg101), a member of ESCRT-I (endosomal sorting complex required for 1

transport I), respectively (14, 16, 17). YPxL and LxxL motifs of EIAV p9 and 2

HIV-1 p6 have been demonstrated to interact with AIP1/Alix, which has also been 3

reported to be linked to ESCRT-I and -III complexes (9, 45). It has been suggested 4

that L-domain motifs may function to recruit their interacting proteins to the sites of 5

virion assembly to facilitate virus egress (5). ESCRT complexes play a critical role 6

in sorting proteins into the multivesicular body (MVB) in mammalian cells (44). In 7

this process, three ESCRT complexes, ESCRT-I, -II, and -III, act in a sequential 8

manner (1, 2). In the final step of protein sorting, an AAA-type ATPase Vps4 9

interacts with ESCRT-III to catalyze the disassembly of ESCRT machinery to recycle 10

its components (3, 4). The expression of dominant negative (DN) forms of and 11

small interfering RNA (siRNA) specific for Tsg101 and Alix/AIP1 inhibit PT/SAP-12

and YPxL-type L-domain-mediated VLP and/or virus release, respectively (9, 12, 14, 13

45). In addition, in many cases, DN forms of Vps4 lacking the ability to bind or 14

hydrolyze ATP were shown to inhibit the budding of VLPs and/or viruses containing 15

any of the PPxY-, PT/SAP-, and YPxL-types of L-domains (12, 14, 32, 45). These 16

observations suggest that viruses possessing these L-domain motifs generally utilize 17

MVB sorting machinery for efficient budding; however, for many other enveloped 18

viruses, L-domain motifs have not yet been identified, and the involvement of MVB 19

sorting machinery in virus budding is still unknown. Recently, in addition to the 20

major L-domain motifs, FPIV and YEIL sequences have been identified as potential 21

L-domain motifs within paramyxovirus SV5 M, and prototype foamy virus (PFV) 22

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Gag proteins, respectively (36, 41). However, the interacting partners of these 1

motifs have not been identified yet (36, 41). In addition, it should be noted that 2

SV5 M protein alone does not have the ability to bud as do VLPs and require other3

viral proteins for efficient budding (42).4

As for Sendai virus (SeV), a prototype of the family Paramyxoviridae, we 5

previously reported that C protein, one of the accessory proteins of SeV, physically 6

interacted with Alix/AIP1, and enhanced VLP budding, possibly depending on the 7

MVB sorting pathway, although C protein did not have the ability to form VLPs (38). 8

Similar to SV5, other viral proteins, such as the nucleoprotein N and two 9

glycoproteins F and HN are required for the integrity of SeV VLP formation; 10

however, matrix (M) protein itself has the ability to be efficiently released from the11

cell surface in the form of VLPs, implying that M protein provides the major driving 12

force for SeV budding, although no known L-domain motifs have been found within 13

M protein, and any links with M protein and host factors are also not known (38, 39, 14

46, 47).15

In this report, we intended to identify an amino acid motif within SeV M protein 16

responsible for efficient M-VLP budding, and to investigate cellular factors involved 17

in this function.18

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Materials and Methods1

Cells and antibodies. Human 293T cells were maintained in Dulbecco’s 2

minimum essential medium (DMEM; Sigma) supplemented with 10% fetal bovine 3

serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel) and 4

penicillin-streptomycin at 37 C. Polyclonal antibodies (pAb) against the whole 5

virion and the C protein of SeV were described previously (46). pAb against GAL4 6

DNA-BD (Sigma) and monoclonal antibody (mAb) against HA-tag (2C16; MBL, 7

Nagoya, Japan) were used according to the protocols of the suppliers.8

Plasmid construction. Plasmids encoding SeV M-WT, C-WT, N-WT, HN-WT, 9

and F-WT in pCAGGS.MCS vector have been described previously (46). M gene 10

mutants (M-A2, M-A4, M-PY, M-PY>A4, M-PT, M-PT>A4, and M-YP) were 11

generated using a standard PCR technique, and inserted into the pCAGGS.MCS 12

vector. A plasmid encoding 5’ HA-tagged AIP1-WT in the pCAGGS.MCS vector 13

has been described previously (38). A series of 5’ HA-tagged AIP1 mutants (1-211, 14

1-423, 1-628, 358-868, 424-628, and 629-868) was generated using a standard PCR 15

technique, and inserted into the pCAGGS.MCS vector. WT and/or mutant genes of 16

M, C, and HA-tagged AIP1 were amplified by PCR, and subcloned into pM GAL4 17

DNA-BD and pVP16 AD cloning vectors (BD Biosciences Clontech) for mammalian 18

two-hybrid assay. All of these constructs were confirmed by DNA sequencing.19

VLP budding assay. Human 293T cells cultured in 6-well plates were20

transfected with the indicated plasmids using FuGENE 6 transfection reagent (Roche 21

Diagnostics). At 24 or 48 hr post-transfection (p.t.), culture medium was harvested 22

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and clarified at 3,000 rpm for 10 min. The supernatant was then centrifuged at 1

40,000 rpm for 2 hr through a 20% sucrose cushion. The pellet was suspended in 2

100 l of SDS-PAGE sample buffer (125 mM Tris-HCl [pH 6.8], 4.6% sodium 3

dodecyl sulfate [SDS], 10% dithiothreitol [DTT], 0.005% bromophenol blue, 20% 4

glycerol), and analyzed by SDS-PAGE (10%) followed by Western blotting using 5

anti-SeV antibody. Cell lysates were also prepared and analyzed by Western 6

blotting using appropriate antibodies. Bands of M proteins were quantitated by 7

densitometry with the ImageJ 1.2.4 program. VLP budding rates were calculated as 8

the ratio of M protein in VLPs to that in cell lysates. For the experiment of Fig. 5, 9

at 24 hr p.t., cells were metabolically labeled with 3.7 MBq/ml of [35S]Met-Cys10

(Pro-mix; Amersham Biosciences) for 24 hr. Culture medium was harvested and 11

clarified at 3,000 rpm for 10 min. The supernatant was then centrifuged at 40,000 12

rpm for 2 hr through a 20% sucrose cushion. The pellet was suspended in 13

radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 1% sodium 14

deoxycholate, 0.1% SDS, 10 mM Tris-HCl [pH 7.4], 150 mM NaCl), 15

immunoprecipitated with anti-SeV pAb, and analyzed by SDS-PAGE. In order to 16

examine the protein expression from transfected plasmids, cell lysates were also 17

immunoprecipitated with the appropriate antibodies, and analyzed by SDS-PAGE. 18

Protein bands were visualized and analyzed with a BAS2000 Bio-imaging analyzer 19

(Fuji Film).20

Pulse-chase experiment. 293T cells cultured in 6-well plates were transfected 21

with the indicated plasmids using FuGENE 6 reagent. At 16 hr p.t., cells were 22

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washed with PBS(-) three times, and incubated with Met-Cys-free medium 1

(Invitrogen) for 30 min. Cells were then metabolically labeled with 3.7 MBq/ml of 2

[35S]Met-Cys for 10 min. Radioactive medium was removed, cells were washed3

with PBS(-), and incubated in fresh growth medium at 37 C. At the indicated time 4

points, cells were suspended in RIPA buffer, and immunoprecipitated with anti-SeV 5

pAb, and analyzed by SDS-PAGE. Protein bands were visualized with a BAS2000 6

Bio-imaging analyzer.7

IP-Western. 293T cells cultured in 6-well plates were co-transfected with the 8

indicated plasmids using FuGENE 6 reagent. At 24 hr p.t., cells were suspended in 9

cell lysis buffer (0.5% NP-40, 20 mM Tris-HCl [pH 7.4], 150 mM NaCl) containing 10

a “complete” protease inhibitor cocktail (Roche Diagnostics). Cell lysate samples 11

were immunoprecipitated with either anti-SeV or -HA antibody. The 12

immunoprecipitates by anti-SeV and -HA antibodies were separated by SDS-PAGE 13

followed by Western blotting using anti-HA and -SeV antibodies, respectively. 14

Protein bands were quantitated as described above. Cell lysates were also subjected 15

directly to Western blotting using anti-HA or -SeV antibody to confirm the 16

expression of Alix/AIP1 and M proteins, respectively.17

Mammalian two-hybrid assay. BD Matchmaker Mammalian Assay Kit 2 (BD 18

Biosciences Clontech) was used for this assay, and experiments were performed 19

according to the protocol of the supplier. GAL4 DNA-BD and VP16 AD plasmids 20

for this assay were prepared as described above. 293T cells were transfected with 21

the indicated AD and BD plasmids together with a pG5SEAP reporter plasmid. At 22

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48 hr p.t., culture medium was harvested and clarified by high-speed centrifugation. 1

Medium samples were applied to fluorescent SEAP assay using BD Great EscAPe 2

SEAP (BD Biosciences Clontech) to quantitate the secreted alkaline phosphatase 3

(SEAP) activity of each sample according to the protocol of the supplier. The 4

fluorescence of each sample was measured using a plate fluorometer (ARVO-SX; 5

Wallac Berthold Japan, Tokyo, Japan).6

siRNA transfection. Synthetic oligonucleotides were inserted between the human 7

U6 promoter and terminator sequence of the pBAsi-hU6 vector (Takara) to generate 8

a stem-loop type of small interfering RNA in transfected cells. pBAsi-AIP1#2147, 9

which targeted nucleotides 2147-CCTAGTGCTCCTTCAATTC-2165 of the AIP1 10

gene, was constructed. A siRNA plasmid for the negative control (pBAsi-NC) was 11

described previously (38). 293T cells in 60-mm dishes were transfected with 1 g12

of the designated pBAsi plasmids. At 24 hr p.t., cells were transfected a second 13

time with 1 g of pCAGGS-M-WT together with 1 g of the pBAsi plasmids. 14

After another 24 hr, cells were metabolically labeled with [35S]Met-Cys for 24 hr, 15

and then analyzed as described above.16

17

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Results1

YLDL sequence within SeV M protein is crucial for VLP budding.2

In order to find an amino acid motif within SeV M protein responsible for VLP 3

formation, we inspected the amino acid sequences of M proteins of paramyxoviruses, 4

such as SeV, human parainfluenza virus type-1 and -3 (HPIV-1 and -3), rinderpest 5

virus (RPV), measles virus (MeV), mumps virus (MuV), phocine distemper virus 6

(PDV), and SV5. Three major L-domain consensus sequences, PS/TAP, PPxY, and 7

YPxL, were not found within any of these M proteins. However, a YxxL motif 8

closely matching the YPxL motif was found in the N-terminus of M proteins of SeV 9

of the genus Respiroviruses, and RPV, PDV, and MeV of the genus Morbilliviruses,10

but not in the M proteins of MuV and SV5 of the genus Rubulaviruses. SeV M 11

protein contains a YLDL sequence starting at amino acid 49, and a trace of this 12

sequence (YLD) starting at amino acids 49 and 53 was found in the M proteins of 13

HPIV-1 and -3 of the genus Respiroviruses, respectively.14

To elucidate the potential function of the YLDL sequence within SeV M protein 15

in VLP budding, we generated two mutants, M-A2 and -A4, in which Y-49 and L-52,16

and the entire YLDL amino acids were replaced by alanines (Fig. 1A). The ability 17

of M-WT, -A2, and -A4 to bud as VLPs was examined using a functional budding 18

assay (Fig. 1). 293T cells were transfected with M-WT, M-A2, and M-A4 plasmids. 19

A striking difference in budding efficiency was observed between M-WT and M 20

mutants (Fig. 1B). M-WT was readily detectable even at 24 hr p.t., and the amount 21

of M-WT released in the culture medium increased with time (Fig. 1B, lanes 2 and 6). 22

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In contrast, the release of M protein into the culture medium was dramatically 1

reduced by amino acid changes within the YLDL sequence, although the amounts of 2

M proteins in cell lysates were not largely different (Fig. 1B, lanes 2 to 4, and 6 to 8). 3

Average quantitation data revealed that the budding efficiencies of M-A2 and M-A4 4

VLPs were approximately 30- and 200-fold reduced compared to that of M-WT, 5

respectively (Fig. 1C). In the pulse-chase experiment, during the chase period up to 6

4 hr, stability of M-A4 was indistinguishable from that of M-WT, indicating that the7

reduced amount of the M mutants released from the transfected cells were not due to 8

the difference in stability between M-WT and the M mutants (Fig. 1D).9

These results indicate that the YLDL sequence within M protein is critical for 10

SeV M-VLP budding.11

The YLDL motif is not functionally replaceable with the major L-domain12

motifs.13

It has been reported that, in most L-domain-identified viruses, L-domain motifs 14

are functionally interchangeable (19, 20, 27, 31, 35, 52). We next sought to 15

determine whether the YLDL sequence within M protein would be functionally 16

replaceable with other types of L-domain motifs. For this purpose, chimeric M 17

proteins (M-PY, and M-PT) were constructed, in which the YLDL motif containing 18

the twelve amino acid region of SeV M protein were replaced by the PPPY 19

motif-containing region from VSV and the PTAP motif-containing region from 20

HIV-1, respectively (Fig. 2A). We also generated two more constructs (M-PY>A4 21

and M-PT>A4) in which the core L-domain motifs of M-PY and -PT were replaced 22

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with four alanines, respectively (Fig. 2A). In addition, we generated an M-YP 1

mutant, in which L-50 was replaced by a proline to possess the YPDL motif from 2

EIAV p9 (Fig. 2A). The ability of these proteins to bud as VLPs was compared to 3

that of M-WT using a functional budding assay as described above. Unexpectedly, 4

unlike many other viruses containing classical L-domain motifs, the release of all 5

these M protein mutants as VLPs was dramatically diminished compared to M-WT, 6

although similar amounts of M protein expression in transfected cells was observed 7

(Fig. 2B). Average quantitation data revealed that the budding efficiency of these 8

chimeric M proteins was more than 100-fold lower than that of M-WT (Fig. 2C). 9

These results indicate that the YLDL sequence of SeV M protein is not functionally 10

replaceable by PPPY, PTAP, and even YPDL-types of L-domains from VSV, HIV-1,11

and EIAV, respectively.12

Alix/AIP1 interacts with SeV M protein in a YLDL motif-dependent manner.13

We next sought to determine whether SeV M protein would interact with 14

Alix/AIP1 in a YLDL sequence-dependent manner, since YPxL and LxxL motifs 15

have been shown to interact with Alix/AIP1 (45).16

At first, we examined the SeV M-Alix/AIP1 interaction by IP-Western (Fig. 3A 17

and 3B). 293T cells were transfected with either M-WT, -A2, -A4, or empty 18

plasmid together with AIP1-WT. At 24 hr p.t., cell lysates were prepared, and 19

immunoprecipitated with the designated antibodies. Immunoprecipitates were 20

separated by SDS-PAGE, and analyzed by Western blotting using the designated 21

antibodies. The amounts of AIP1-WT and M proteins in the transfected cells were 22

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not largely different, respectively (Fig. 3A, lanes 15 to 18, 20, and 22 to 24). 1

AIP1-WT was co-immunoprecipitated with M-WT, but the levels of AIP1-WT 2

co-immunoprecipitated with M-A2 and -A4 were reduced by an average of 2.5- and 3

24-fold compared to that with M-WT (Fig. 3A, lanes 4 to 5, and Fig. 3B). Similarly, 4

M-WT was co-immunoprecipitated with AIP1-WT, but M-A2 and -A4 were not (Fig. 5

3A, lanes 10 to 12).6

We further examined the interaction between SeV M and Alix/AIP1 using a 7

mammalian two-hybrid technique (Fig. 3C and 3D). 293T cells were co-transfected8

with M-WT, -A2, or -A4 fused with GAL4 DNA binding domain (BD), and 9

AIP1-WT fused with the VP16 activation domain (AD) (Fig. 3C). Similarly, cells 10

were co-transfected with AD-fused M protein mutants and BD-fused AIP1-WT (Fig. 11

3D). Almost equivalent amounts of AIP1-WT and M proteins were expressed in12

the transfected cells, respectively (Fig. 3C and 3D, lanes 3 to 6 and 10 to 12). Bar 13

graphs of average SEAP activity showed the interaction of AIP1-WT with M-WT, 14

but SEAP activity was reduced in M-A2 or -A4-transfected samples (Fig. 3C and 15

3D).16

These results indicate that Alix/AIP1 physically interacts with SeV M protein in a 17

YLDL sequence-dependent manner.18

N-terminal 211 amino acids of Alix/AIP1 are important for interaction with M 19

protein.20

To investigate which region of Alix/AIP1 is required for interaction with M 21

protein, a series of deletion mutants of Alix/AIP1 was generated (Fig. 4A). 293T 22

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cells were co-transfected with BD-fused M-WT and a series of AD-fused Alix/AIP1 1

mutants together with the SEAP reporter plasmid, and SEAP activity at 48 hr p.t. was 2

measured. Almost equivalent amounts of M-WT were expressed in transfected 3

cells, and the expression of each Alix/AIP1 mutant was confirmed (Fig. 4B). Bar 4

graphs of average SEAP activity showed that AIP1-WT as well as Alix/AIP1 mutants, 5

AIP1(1-211), AIP1(1-423), and AIP1(1-628), containing N-terminal Bro-like domain, 6

interacted with M-WT, but other mutants, AIP1(358-868), AIP1(424-628) and 7

AIP1(628-868), which did not contain the domain, did not (Fig. 4C). This result 8

indicates that the N-terminal region of Alix/AIP1 at positions of 1 to 211, including 9

the Bro-like domain, is important for interaction with SeV M protein.10

Alix/AIP1 functions in SeV M-VLP budding.11

To investigate whether Alix/AIP1 would function in the budding of SeV M-VLPs, 12

we first examined whether overexpression of the deletion mutants of Alix/AIP1 13

could affect the efficiency of M-VLP budding (Fig. 5). 293T cells were 14

co-transfected with M-WT and a series of Alix/AIP1 mutants. At 24 hr p.t., cells 15

were metabolically labeled with [35S]Met-Cys for 24 hr. VLP samples and cell 16

lysates were harvested, immunoprecipitated with anti-HA mAb or anti-SeV pAb, and 17

analyzed by SDS-PAGE followed by autoradiography. Almost identical amounts of 18

M-WT were expressed in transfected cells (Fig. 5A, lanes 9 to 16), and the 19

expression of Alix/AIP1 mutants was confirmed (Fig. 5A, lanes 17 to 23). 20

Quantitation of radioactivity revealed that the amount of M-WT in VLPs hardly 21

changed in the presence of AIP1-WT as well as most Alix/AIP1 mutants, whereas22

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only approximately 20% and 40% by overexpression of AIP1(424-628) containing 1

two C-C domains, and AIP1(1-211) containing the N-terminal Bro-like domain, 2

respectively, compared to the empty vector-transfected sample (Fig. 5).3

Furthermore, we examined the effect of the depletion of endogenous Alix/AIP1 4

by a specific siRNA, AIP1#2147, on the budding of M-VLPs (Fig. 6A). The 5

expression of Alix/AIP1 from the transfected plasmid was efficiently inhibited in 6

cells receiving AIP1#2147 (Fig. 6B). Identical protein profiles of total protein 7

extracts from non-specific (NS) siRNA- and AIP1#2147-transfected cells were 8

observed following staining with Coomassie blue (Fig. 6C). The amount of M-WT 9

in VLPs was reduced by more than 90% in AIP1#2147-transfected cells, compared 10

to that in NS siRNA-transfected cells (Fig. 6A, lanes 1 and 2, and bar graph), 11

although the amounts of M-WT expressed in transfected cells were not largely 12

different (Fig. 6A, lanes 3 and 4).13

These results suggest that Alix/AIP1 is functionally involved in SeV M-VLP 14

budding.15

Alix/AIP1 interacts with SeV C and M proteins in different regions. 16

Since we previously reported that SeV C protein also physically interacted with 17

Alix/AIP1 (38), we further investigated which region of Alix/AIP1 was required for 18

interaction with C protein (Fig. 7). 293T cells were co-transfected with BD-fused19

C-WT and a series of AD-fused Alix/AIP1 mutants together with the SEAP reporter 20

plasmid, and SEAP activity at 48 hr p.t. was determined. Similar amounts of C-WT 21

were expressed in transfected cells, and the expression of each Alix/AIP1 mutant was 22

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confirmed (Fig. 7A). The bar graph of average SEAP activity showed that, like 1

SeV M protein, C-WT interacted with AIP1-WT as well as two N-terminal mutants 2

AIP1(1-423) and AIP1(1-628) but, unlike M protein, not with the N-terminal mutant 3

AIP1(1-211) as well as mutants AIP1(358-868), AIP1(424-628), and AIP1(628-868)4

(Fig. 7B). This result indicates that, unlike M protein, a SeV C-binding site is 5

located between amino acid positions 212-357 of Alix/AIP1.6

Enhancement of the budding of M-VLPs by C protein is independent of the 7

presence of the YLDL sequence.8

Finally, we investigated whether C protein would enhance the budding of VLPs 9

of M-WT and M-A4 in the absence of other viral proteins (Fig. 8), because we 10

previously reported that SeV C protein enhanced the budding efficiency of SeV 11

M-VLPs in the presence of additional viral proteins, N, F, and HN, required for the 12

integrity of virion formation (38). 293T cells were co-transfected with the plasmids 13

indicated in the figure. At 48 hr p.t., VLP samples and cell lysates were prepared 14

and analyzed by SDS-PAGE followed by Western blotting using anti-SeV and anti-C15

pAbs. As expected from the previous report that N protein was released into 16

culture medium in the form of a nucleocapsid-like structure together with cellular 17

RNAs (46), N protein was readily detectable in VLP samples from N 18

gene-transfected cells regardless of the amount of M proteins released (Fig. 8A, lanes 19

3, 4, 7, and 8). The expression levels of each transfected protein in cells were not 20

largely different (Fig. 8B). Consistent with our previous report (38), VLP budding 21

of M-WT was more than 2-fold increased in the presence of C-WT as well as 22

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additional viral proteins (Fig. 8A, lanes 3 and 4, and Fig. 8C). C-WT alone also 1

enhanced VLP budding of M-WT approximately 2-fold (Fig. 8A, lanes 1 and 2, and 2

Fig. 8C). M-A4 in VLPs was not detectable in the absence of additional N, F, and3

HN proteins due to its budding deficiency (Fig. 8A, lanes 5 and 6, and Fig. 8C), 4

whereas approximately 5-fold enhancement of M-A4 in VLP by C-WT was observed 5

in the presence of additional proteins (Fig. 8A, lanes 7 and 8, and Fig. 8C). This 6

result indicates that SeV C protein enhances M-VLP budding independently of 7

M-Alix/AIP1 interaction regardless of the presence or absence of additional viral 8

proteins, N, F, and HN.9

10

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Discussion1

The budding of enveloped RNA viruses has been extensively studied, particularly2

for retroviruses, since three major L-domain motifs, PPxY, PT/SAP, and YPxL were 3

identified within Gag proteins. Common features among L-domains have been 4

reported. L-domain motifs are functionally interchangeable (19, 20, 27, 31, 35, 52),5

and the budding of retroviruses possessing any of the three major types of L-domain6

motifs is inhibited by overexpression of DN forms of Vps4 (12, 14, 32, 45). In 7

addition, cellular factors, such as Nedd4, Tsg101, and Alix/AIP1, which have been 8

identified as interacting partners for PPxY, PT/SAP, and YPxL types of L-domain9

motifs, respectively, are components of ESCRT complexes and/or have shown some 10

link to MVB sorting machinery (8, 32, 50). From these observations, most 11

enveloped viruses are believed to commonly utilize cellular MVB sorting machinery 12

for efficient budding.13

In contrast, important functional differences of viral L-domains have also been 14

reported. L-domains have been shown to function in a cell-type-dependent manner 15

(13), and not all L-domains are functionally interchangeable (30, 51, 52). Unlike 16

PPxY-type L-domain-containing retroviruses, budding of the PPxY motif containing 17

rhabdoviruses and that of an Ebola VP40 mutant VP40-dPTA, in which only the 18

PPEY motif was retained but the PTAP motif was abolished by deleting the first 19

three PTA amino acids of the overlapping motifs, was not sensitive to DN forms of 20

Vps4 (21). In addition, for many enveloped viruses, no L-domain motifs have been 21

identified yet, and the involvement of MVB sorting machinery in their budding is 22

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still unknown. As for paramyxoviruses, L-domain motifs have not been identified 1

yet except for SV5, whose FPIV motif within M protein is reported to act as an 2

L-domain (41).3

In this report, we first found that the YLDL sequence located at the N-terminus of 4

M protein was critical for SeV M-VLP budding. Mutations in this sequence 5

dramatically reduced the efficiency of M-VLP budding (Fig. 1). It is unclear 6

whether the reduced VLP budding is due to budding defect, impaired VLP assembly, 7

disruption of M protein trafficking, or other defects in the VLP assembly and release 8

pathway. Experiments to determine the mechanism of the reduced budding is9

currently under way. The YxxL sequence was also found within M proteins of 10

other paramyxoviruses, such as RPV, PDV, and MeV belonging to Morbilliviruses at 11

similar positions to the YLDL motif of SeV M, whereas Rubulaviruses, such as MuV 12

and SV5, did not contain a YxxL motif as well as other known L-domain motifs 13

within M proteins, implying that the budding mechanism of Respiroviruses and14

Morbilliviruses may be different from that of Rubulaviruses.15

We also found that SeV M protein physically interacts with Alix/AIP1 via the 16

YLDL sequence (Figs. 3 and 4). M-VLP budding was inhibited by the 17

overexpression of two deletion mutants of Alix/AIP1, AIP1(1-211) and 18

AIP1(424-628), and by the depletion of endogenous Alix/AIP1 using RNAi 19

technique (Figs. 5 and 6), suggesting that Alix/AIP1 is functionally involved in SeV 20

M-VLP budding. Since it contains a SeV M binding site, the AIP1(1-211) mutant 21

may interfere with the interaction between intact Alix/AIP1 and the M proteins. 22

- 20 -

Overexpression of DN forms of the components of endosomal sorting machinery, 1

such as Vps4, CHMP4B, and Tsg101 induces formation of aberrantly enlarged 2

endosomes (called “class E” compartment in yeast) that are defective in the sorting 3

and recycling of endocytosed substrates (6, 7, 18). AIP1(1-211) and/or 4

AIP1(424-628) not containing a SeV M binding site may inhibit budding by 5

interfering with some functions of Alix/AIP1, which are utilized for the budding 6

process. Inconsistent with our previous report that overexpression of Alix/AIP1 7

accelerated SeV release from the infected cells (38), budding of M-VLP was not 8

enhanced by Alix/AIP1 overexpression (Fig. 5). This difference seems likely due to 9

the presence or absence of the other viral proteins. As expected, no interaction of 10

SeV M with Tsg101 and Nedd4, which have been known to interact with PT/SAP 11

and PPxY types of L-domain motifs, and no inhibitory effect on M-VLP budding by 12

DN forms of Tsg101 and Nedd4 were observed (data not shown).13

In contrast to SeV, the FPIV motif within SV5 M protein is reported not to 14

interact with Alix/AIP1, although it resembles the YPxL motif that interacts with 15

Alix/AIP1 (41). Recently, another YxxL-type motif, YEIL, has also been identified 16

as a potential L-domain within the Gag protein of prototype foamy virus (36). The 17

YEIL motif is also reported not to interact with Alix/AIP1, although its binding 18

partner has not been identified yet (36). Unlike SeV, it has been reported that a 19

single expression of SV5 M protein is not enough to form VLPs, and that multiple20

viral proteins are required for the formation and budding of VLPs (42). These21

differences between SeV and SV5 may reflect their different mechanism in budding. 22

- 21 -

In spite of the functional differences of the L-domain motif of SeV M from those of 1

other viruses, all these viruses also seem to utilize MVB sorting machinery for 2

efficient budding, since DN forms of Vps4 block VLP and/or virus budding of all 3

these viruses (36, 38, 42).4

In addition to SV5, the contribution of multiple viral proteins for efficient VLP 5

budding has been reported in some enveloped viruses. It is known that VLP 6

budding by a matrix protein is enhanced in the presence of additional viral proteins, 7

such as VSV G, Ebola virus GP and NP, although the mechanism for such 8

enhancement remains to be determined (29, 37, 43). We previously reported that9

SeV C protein enhanced SeV M-VLP budding in the presence of additional viral N, F, 10

and HN proteins, and interaction between C and Alix/AIP1 correlated with this 11

enhancement (38). Interestingly, we found that SeV M protein also physically 12

interact with Alix/AIP1 at the distinct region on Alix/AIP1 from C protein (amino 13

acid positions 1-211 for M, and 212-357 for C) (Figs. 4 and 7). The C protein does 14

not have any known amino acid motifs responsible for interaction with Alix/AIP1. 15

Chen et al. demonstrated that the EIAV YPDL motif interacted with a region of 16

Alix/AIP1 spanning amino acids 409-715 (9). This discrepancy might reflect 17

differences in the experimental system used, or differences in L-domain function 18

between the SeV YLDL and the EIAV YPDL motifs. Interaction between SeV M 19

and Alix/AIP1 seems more important for VLP budding than that between C protein 20

and Alix/AIP1, because C protein itself does not have the ability to be released from 21

cells, and budding of SeV M-VLP was dramatically inhibited by mutations in the 22

- 22 -

YLDL motif causing loss of M-Alix/AIP1 interaction. Our results also showed that 1

C protein could enhance SeV M-VLP budding regardless of the presence or absence 2

of other viral proteins and independently of M-Alix/AIP1 interaction (Fig. 8). From 3

these results, SeV M protein is by itself able to bud from the cell surface in the form 4

of VLPs by interacting with Alix/AIP1, whereas SeV C protein may provide more 5

Alix/AIP1 by C-Alix/AIP1 interaction and enhance efficiency to utilize cellular 6

MVB sorting machinery for efficient VLP and/or virus budding. It will be interest 7

to elucidate the mechanism of how SeV M and C proteins utilize Alix/AIP1 functions 8

and cooperate with each other for efficient budding. Recently, HIV-1 Nef has been 9

reported to interact with Alix/AIP1, resulting in the proliferation of multivesicular 10

bodies and enhancement of budding efficiency, in addition to the interaction of Gag 11

with Tsg101 and Alix/AIP1 via its PTAP and LxxL motifs, respectively (10). 12

Alix/AIP1 is not only a component of MVB sorting machinery, but also has the 13

ability to generate an MVB-resembling membrane structure in vitro in the presence 14

of a specific phospholipid, lysobisphosphatidic acid, and to form exosomes, small 15

membrane structures that are liberated from cells (33, 48). Such functions of 16

Alix/AIP1 itself may contribute to increase the efficiency of virus budding in 17

addition to MVB sorting machinery.18

Finally, the SeV YLDL motif is similar to the EIAV YPDL motif, in terms of 19

their importance in VLP budding, closely matching amino acid sequences, and 20

ability to interact with Alix/AIP1. However, important differences are also 21

observed. The YLDL motif is unique in not containing a proline residue, different 22

- 23 -

from most other L-domain motifs. The SeV YLDL motif-binding site was mapped 1

to a region of Alix/AIP1 (amino acid positions 1-211), whereas the EIAV YPDL 2

motif interacted with a different region of Alix/AIP1 (amino acid positions 409-715),3

as mentioned above (9). Surprisingly, the YLDL motif was not functionally4

replaceable with even the EIAV YPDL motif as well as the VSV PPPY and HIV-15

PTAP motifs (Fig. 2). These findings might imply fundamental differences in the 6

budding mechanisms mediated by the SeV YLDL motif and the other characterized 7

L-domain motifs.8

9

- 24 -

Acknowledgements1

We would like to thank the staff of the Research Center for Molecular Medicine 2

and the Analysis Center of Life Science, Hiroshima University for the use of their 3

facilities. This work was supported by Grants-in-Aid for Scientific Research from 4

the Japan Society of the Promotion of Science and the Ministry of Education, Culture, 5

Sports, Science and Technology.6

7

8

- 25 -

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13

14

15

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Figure Legends1

Fig. 1. Budding assay of SeV M proteins. (A) Schematic representation of 2

expression plasmids encoding M-WT, -A2, and -A4. (B) M proteins were 3

expressed in 293T cells. At the indicated time points, cell lysates and VLPs in 4

culture medium were harvested, and analyzed by SDS-PAGE followed by Western 5

blotting using anti-SeV pAb. (C) M proteins in VLPs and cell lysates were 6

quantitated by densitometry with ImageJ software. The values of M protein in 7

VLPs were normalized by those in cell lysates. The level of M-WT at each time 8

point was set to 1, and the relative values of M-A2 and -A4 are indicated. Bars 9

represent an average of at least three independent experiments. Error bars indicate 10

standard deviations. (D) Pulse-chase experiments for M-WT and M-A4. 293T 11

cells transfected with M-WT and M-A4 were pulse-labeled with [35S]Met-Cys for 10 12

min. At the indicated time points, cell lysates were prepared, immunoprecipitated 13

by anti-SeV pAb, and analyzed by SDS-PAGE. Protein bands were visualized with 14

a BAS2000 Bio-imaging analyzer.15

Fig. 2. Budding assay of SeV M mutants possessing the VSV PPPY and HIV-116

PTAP motif-containing regions or the EIAV YPDL motif. (A) The amino acid 17

sequence within the YLDL motif-containing region (amino acids 45 to 56) is shown 18

for M-WT, -PY, -PY>A4, -PT, -PT>A4, and -YP as shown in Fig. 1. (B and C) 19

These mutants were subjected to a functional budding assay, and results are shown in 20

Fig. 1. The bar graph represents an average of at least three independent 21

experiments.22

- 34 -

Fig. 3. Interaction between SeV M and Alix/AIP1. (A) IP-Western study for M 1

mutants and Alix/AIP1. 293T cells were co-transfected with the indicated plasmids. 2

At 24 hr p.t., cell lysates were harvested, and immunoprecipitated by the indicated3

Abs. The immunoprecipitates were then analyzed by Western blotting using the 4

indicated Abs. (B) Alix/AIP1 protein immunoprecipitated with anti-SeV pAb and 5

M proteins in cell lysates was quantitated by densitometry with ImageJ software. 6

The level of Alix/AIP1 from the sample co-transfected with M-WT was set to 1, and 7

the relative values of Alix/AIP1 were normalized by those of M proteins in cell 8

lysates. Bars represent an average of three independent experiments. (C and D) 9

Mammalian two-hybrid analysis for M mutants and AIP1-WT. SeV M mutants and 10

AIP1-WT were subcloned into pVP16 (AD) and pM (BD) vectors. 293T cells were 11

co-transfected with the indicated AD and BD plasmids together with a pG5SEAP 12

reporter plasmid. At 48 hr p.t., SEAP activity in the culture medium was 13

determined using a fluorometer. Bars represent an average of three independent 14

experiments. Expressions of Alix/AIP1 and M mutants were confirmed by Western 15

blotting using anti-HA mAb for Alix/AIP1 and anti-SeV pAb for M mutants.16

Fig. 4. Mammalian two-hybrid analysis for M-WT and a series of deletion mutants 17

of Alix/AIP1. (A) Schematic representation of AIP1-WT and the Alix/AIP1 18

deletion mutants used in this study. Bro-like domain, Coiled-Coil domain, and 19

Pro-rich domain reported by Katoh et al. (26) are highlighted in gray. (B and C) 20

293T cells were co-transfected with BD-fused M-WT and the indicated AD plasmids 21

together with a SEAP reporter plasmid. Expressions of M protein and Alix/AIP1 22

- 35 -

mutants were confirmed by Western blotting as in Fig. 3. SEAP activity in the 1

culture medium was determined using a fluorometer at 48 hr p.t. Bars represent an 2

average of three independent experiments.3

Fig. 5. Effect of overexpression of Alix/AIP1 mutants on budding of SeV M-VLPs. 4

(A) 293T cells were co-transfected with M-WT and the indicated Alix/AIP1 mutants. 5

At 24 hr p.t., cells were radio-labeled for another 24 hr. Cell lysates and VLPs were6

immunoprecipitated with anti-SeV pAb for M protein and anti-HA mAb for 7

Alix/AIP1 mutants. (B) M proteins present in VLPs and cell lysates were 8

quantitated. The values of M proteins in VLPs were normalized by those in cell 9

lysates, and the levels of M protein in VLPs from cells transfected with an empty 10

vector, (-), was set to 1. Bars represent an average of three independent 11

experiments.12

Fig. 6. Effect of Alix/AIP1 depletion on budding of SeV M-VLP. (A) 293T cells 13

were transfected with Alix/AIP1-specific (#2147) or non-specific (NS) siRNA 14

plasmids. At 24 hr p.t., cells were further co-transfected with M-WT and siRNA 15

plasmids, and radio-labeled. Immunoprecipitation of M protein from VLPs (lanes 1 16

and 2) and cell lysates (lanes 3 and 4) are indicated. M proteins in VLPs and cell 17

lysates were quantitated. The values of M proteins in VLPs were normalized by 18

those in cell lysates. The level of M protein in VLPs from NS siRNA 19

plasmid-transfected cells was set to 1. Bars represent an average of three20

independent experiments. (B) Western blot demonstrating that Alix/AIP1 from the 21

transfected plasmid is inhibited following transfection of AIP1#2147, but is not 22

- 36 -

inhibited following transfection of NS-siRNA. (C) Coomassie blue stain of total 1

protein extract from NS-siRNA transfected cells and AIP1#2147-transfected cells.2

Fig. 7. Mammalian two-hybrid analysis for C-WT and a series of deletion mutants 3

of Alix/AIP1. 293T cells were co-transfected with BD-fused C-WT and the 4

indicated AD plasmids together with a SEAP reporter plasmid. (A) Expressions of 5

C protein and Alix/AIP1 mutants were confirmed by Western blotting using anti-BD6

mAb for C-WT and anti-HA mAb for Alix/AIP1 mutants. (B) SEAP activity in the 7

culture medium was determined using a fluorometer at 48 hr p.t. Bars represent an 8

average of three independent experiments.9

Fig. 8. Budding assay of M-WT and M-A4 in the presence or absence of other viral 10

proteins. 293T cells were co-transfected with the indicated plasmids. At 48 hr p.t., 11

VLPs (A) in the culture medium and cell lysates (B) were harvested, and analyzed by 12

SDS-PAGE followed by Western blotting using anti-SeV and -C pAbs. (C) M 13

proteins in VLPs and cell lysates were quantitated by densitometry with ImageJ 14

software. The values of M protein in VLPs were normalized by those in cell lysates. 15

The level of M-WT from cells not transfected with any additional viral proteins, (-),16

was set to 1. Bars represent an average of three independent experiments.17

18

Fig. 1. Irie et al.

SeV M

(A)

(C)

(D)

1 2 3 4 5 6 7 8

M -

M -

(-) M-WT

M-A2

M-A4

(-) M-WT

M-A2

M-A4

24 h 48 h

M -

(B) VLPs

Cells

(min.) 0 30 60 120 240 0 30 60 120 240M-WT M-A4

(B)

(C)

Fig. 2. Irie et al.

SeV M

(A)

M -

9 10 11 12 13

(-) M-WT

M-WT

M-YP

M-PY

M-PY>A4

M-PT

M-PT>A4

M -2 3 4 51 6 7 8

14

VLPs

Cells

15 16

Fig. 3. Irie et al.

(A)

(B)

M-WT

M-A2

M-A4

(-) M-WT

(-)

AIP1-WT +:

AIP1 -

2 3 4 51 6

M -

8 9 10 117 12

AIP1 -

14 15 16 1713 18

M -

19 20 21 22 23 24

IP: anti-SeV, WB: anti-HA

IP: anti-HA, WB: anti-SeV

WB: anti-HA

WB: anti-SeV

(C)

AD + BD

BD BD-M-WT

BD-M-A2

BD-M-A4

(-)

AD-AIP1-WT +:

AIP1 -

M -

2 3 4 51

7 8 9 10 11

6

12

(D)

AD + BD

AD

AD-M-WT

AD-M-A2

AD-M-A4

(-)

BD-AIP1-WT +:

AIP1 -

M -

2 3 4 51

7 8 9 10 11

6

12

Fig. 4. Irie et al.

(A)

Coiled-Coil domain

Bro-like domain Pro-rich domainAIP1-WT

1-211

1-423

1-628

358-868

424-628

629-868

(B)

M -

(-) AD + BD

AD

AD-AIP1-WT

AD-AIP1(1-211)

AD-AIP1(1-423)

AD-AIP1(1-628)

AD-AIP1(358-868)

AD-AIP1(424-628)

AD-AIP1(629-868)

BD-M-WT +:

AIP1 mutants

*

*

*

* *

**

(C)

Fig. 5. Irie et al.

(A)

(B)

M -

AIP1 mutants

9 10 11 12 13 14 15

17 18 19 20 21 22 23

16

AIP1(358-868)

AIP1(629-868)

AIP1(424-628)

AIP1(1-211)

AIP1(1-423)

AIP1(1-628)

AIP1-WT

(-)

M -1 2 3 4 5 6 7 8

VLPs

Cells

Fig. 6. Irie et al.

AIP1#2147

NS

AIP1#2147

NS

M -

M -

AIP1 -1 2

3 4

Cells

VLPs

AIP1#2147

NS

(A) (B) (C)

Fig. 7. Irie et al.

(A)

(B)

C -

(-) AD + BD

AD

AD-AIP1-WT

AD-AIP1(1-211)

AD-AIP1(1-423)

AD-AIP1(1-628)

AD-AIP1(358-868)

AD-AIP1(424-628)

AD-AIP1(629-868)

BD-C-WT +:

AIP1 mutants

**

**

*

*

*

Fig. 8. Irie et al.

(A) VLPsM-WT +: M-A4 +:

C

N + F + HN

N + F + HN + C

(-)

C

N + F + HN

F - HN - N

M -

1 2 3 4 5 6 7 8

N + F + HN + C

(-)

(B) Cells

C -

F -HN -N

M -

9 10 11 12 13 14 15 16

M-WT +: M-A4 +:

C N + F + HN

N + F + HN + C

(-) C N + F + HN

N + F + HN + C

(-)

(C)

0

0.5

1

1.5

2

2.5

Rela

tive

dens

ity o

f M p

rote

in

(-) C

N+F+HN

N+F+HN+C (-) C

N+F+HN

N+F+HN+C

M-WT+: M-A4+: