The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein

Abstract

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.

FIG. 1.

Budding assay of SeV M proteins. (A) Schematic representation of expression plasmids encoding M-WT, -A2, and -A4. (B) M proteins were expressed in 293T cells. At the indicated time points, cell lysates and VLPs in culture medium were harvested and analyzed by SDS-PAGE, followed by Western blotting with anti-SeV PAb. (C) M proteins in VLPs and cell lysates were quantitated by densitometry with ImageJ software. The concentrations of M protein in VLPs were normalized to those in cell lysates. The level of M-WT at each time point was set to 1, and the relative values of M-A2 and M-A4 are indicated. Bars represent averages of at least three independent experiments. Error bars indicate standard deviations. (D) Pulse-chase experiments for M-WT and M-A4. 293T cells transfected with M-WT and M-A4 were pulse-labeled with [³⁵S]Met-Cys for 10 min. At the indicated time points, cell lysates were prepared, immunoprecipitated by anti-SeV PAb, and analyzed by SDS-PAGE. Protein bands were visualized with a BAS2000 bioimaging analyzer.

FIG. 2.

Budding assay of SeV M mutants possessing the VSV PPPY and HIV-1 PTAP motif-containing regions or the EIAV YPDL motif. (A) The amino acid sequence within the YLDL motif-containing region (amino acids 45 to 56) is shown for M-WT, M-PY, M-PY>A4, -MPT, M-PT>A4, and M-YP as shown in Fig. 1. (B and C) These mutants were subjected to a functional budding assay, and results are shown in Fig. 1. The bar graph represents an average of at least three independent experiments.

FIG. 3.

Interaction between SeV M and Alix/AIP1. (A) IP-Western blotting (WB) of M mutants and Alix/AIP1. 293T cells were cotransfected with the indicated plasmids. At 24 hpt, cell lysates were harvested and immunoprecipitated by the indicated antibodies. The immunoprecipitates were then analyzed by Western blotting with the indicated antibodies. (B) Alix/AIP1 protein immunoprecipitated with anti-SeV PAb and M proteins in cell lysates were quantitated by densitometry with ImageJ software. The level of Alix/AIP1 from the sample cotransfected with M-WT was set to 1, and the relative values of Alix/AIP1 were normalized to those of M proteins in cell lysates. Bars represent an average of three independent experiments. (C and D) Mammalian two-hybrid analysis for M mutants and AIP1-WT. SeV M mutants and AIP1-WT were subcloned into pVP16 (AD) and pM (BD) vectors. 293T cells were cotransfected with the indicated AD and BD plasmids together with a pG5SEAP reporter plasmid. At 48 hpt, SEAP activity in the culture medium was determined with a fluorometer. Bars represent an average of three independent experiments. Expression of Alix/AIP1 and M mutants was confirmed by Western blotting with anti-HA MAb for Alix/AIP1 and anti-SeV PAb for M mutants.

FIG. 4.

Mammalian two-hybrid analysis for M-WT and a series of deletion mutants of Alix/AIP1. (A) Schematic representation of AIP1-WT and the Alix/AIP1 deletion mutants used in this study. The Bro-like domain, coiled-coil domain, and Pro-rich domain reported by Katoh et al. (26) are highlighted in gray. (B and C) 293T cells were cotransfected with BD-fused M-WT and the indicated AD plasmids together with a SEAP reporter plasmid. Expression of M protein and Alix/AIP1 mutants was confirmed by Western blotting as described in the legend to Fig. 3. SEAP activity in the culture medium was determined with a fluorometer at 48 hpt. Bars represent averages of three independent experiments.

FIG. 5.

Effect of overexpression of Alix/AIP1 mutants on budding of SeV M-VLPs. (A) 293T cells were cotransfected with M-WT and the indicated Alix/AIP1 mutants. At 24 hpt, cells were radiolabeled for another 24 h. Cell lysates and VLPs were immunoprecipitated with anti-SeV PAb for M protein and anti-HA MAb for Alix/AIP1 mutants. (B) M proteins present in VLPs and cell lysates were quantitated. The values of M proteins in VLPs were normalized to those in cell lysates, and the levels of M protein in VLPs from cells transfected with an empty vector [(−)] was set to 1. Bars represent averages of three independent experiments.

FIG. 6.

Effect of Alix/AIP1 depletion on budding of SeV M-VLP. (A) 293T cells were transfected with Alix/AIP1-specific (AIP1#2147) or NS siRNA plasmids. At 24 hpt, cells were further cotransfected with M-WT and siRNA plasmids and radiolabeled. Immunoprecipitation of M protein from VLPs (lanes 1 and 2) and cell lysates (lanes 3 and 4) is indicated. M protein levels in VLPs and cell lysates were quantitated. The M protein concentrations in VLPs were normalized to those in cell lysates. The level of M protein in VLPs from NS siRNA plasmid-transfected cells was set to 1. Bars represent averages of three independent experiments. (B) Western blot demonstrating that Alix/AIP1 from the transfected plasmid is inhibited following transfection of AIP1#2147 but is not inhibited following transfection of NS-siRNA. (C) Coomassie blue staining of total protein extract from NS-siRNA-transfected cells and AIP1#2147-transfected cells.

FIG. 7.

Mammalian two-hybrid analysis for C-WT and a series of deletion mutants of Alix/AIP1. 293T cells were cotransfected with BD-fused C-WT and the indicated AD plasmids together with a SEAP reporter plasmid. (A) Expression of C protein and Alix/AIP1 mutants was confirmed by Western blotting with anti-BD MAb for C-WT and anti-HA MAb for Alix/AIP1 mutants. (B) SEAP activity in the culture medium was determined with a fluorometer at 48 hpt. Bars represent averages of three independent experiments.

FIG. 8.

Budding assay of M-WT and M-A4 in the presence or absence of other viral proteins. 293T cells were cotransfected with the indicated plasmids. At 48 hpt, VLPs in the culture medium (A) and cell lysates (B) were harvested and analyzed by SDS-PAGE, followed by Western blotting with anti-SeV and anti-C PAb. (C) M proteins in VLPs and cell lysates were quantitated by densitometry with ImageJ software. The values of M protein in VLPs were normalized to those in cell lysates. The level of M-WT from cells not transfected with any additional viral proteins, (−), was set to 1. Bars represent averages of three independent experiments.