FIG 6.

Ubiquitin regulation and appropriate membrane targeting of the key Leu302 residue in NES is critical for M protein VLP budding. (A) The L302A mutant is deficient in VLP budding. L302 and L305 in the HPIV3 M protein were mutated to alanines, and a VLP budding assay of the two mutants was performed as described in the legend to Fig. 1. WB was performed with anti-HA Ab. Quantification of the budding index for the wild-type and mutant M proteins is shown. Standard errors were calculated from three independent experiments. The size markers represent a dimer and tetramer of M in VLPs. (B) Ubiquitination of wild-type M and mutants L302A and L305A. 293T cells were transfected with the indicated plasmids, and a coimmunoprecipitation assay was performed as described in the legend to Fig. 3. (C) Cellular localization of wild-type M and mutants L302A and L305A. HeLa cells were transfected with the indicated plasmids. At 24 h posttransfection, cells were fixed and stained with anti-HA Ab and visualized via confocal microscopy as described in Materials and Methods. (D) L302A is defective in membrane association compared to wild-type M and L305A. 293T cells expressing wild-type M/eGFP, L302A/eGFP, or L305A/eGFP were harvested at 48 h posttransfection. Cell homogenates were subjected to a membrane association assay as described in Materials and Methods. Membrane-associated proteins mainly exist in fraction 2. Endogenous calnexin and exogenous eGFP proteins were included as positive controls for membrane-associated and non-membrane-associated proteins, respectively. (E) Fusion of membrane-targeting signals can resolve the budding defect of L302A. L10 and S15 were fused to the N terminus of either L302A or H1N1-M1. 293T cells were transfected with the indicated constructs, and a VLP budding assay for these fusing proteins was performed as described in the legend to Fig. 1. Quantification of the budding index for the corresponding protein is shown. Standard errors were calculated from three independent experiments.