Mutation of YMYL in the Nipah virus matrix protein abrogates budding and alters subcellular localization

Abstract

Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.

FIG. 1.

NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in radioimmunoprecipitation assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.

FIG. 2.

Budding of NiV VLPs is dependent on the presence YMYL. (a) Illustration of NiV M YMYL mutant constructs. WT, wild type. (b) HEK 293T cells were transfected with the indicated expression plasmids, and at 48 hours posttransfection, VLPs were purified as described in the legend to Fig. 1. Western blotting (IB) was performed with anti-GFP and anti-HA antibodies. Two sections of the same blot after the same exposure time are shown.

FIG. 3.

Localization of wild-type and YMYL mutant NiV M proteins in transfected cells. (a) MDCK and 293T cells were transfected with plasmids expressing the indicated wild-type (WT) or mutant NiV M proteins tagged at the amino terminus with HA epitope (for the MDCK cells) or with GFP (for the 293T cells). At 24 hours posttransfection, the transfected MDCK cells were fixed and stained for immunofluorescence microscopy with anti-HA monoclonal antibody (red), and the cell nuclei were stained with Hoechst nuclear dye (green). The 293T cells were fixed at 24 hours posttransfection and imaged for GFP. (b) Quantification of the localization of wild-type and mutant NiV M in MDCK cells (100 fixed, anti-HA antibody-stained cells positive for NiV M were counted per transfection).

FIG. 4.

NiV M wild-type (WT) and YMYL mutant proteins self-associate. 293T cells were transfected as indicated with FLAG- or HA-tagged versions of the constructs shown in Fig. 2a. Immunoprecipitation (IP) of all lysates was done with anti-FLAG beads, followed by Western blotting (IB) with anti-HA antibody. FLAG and HA blots of whole-cell extract (WCE) are shown in the bottom panel.

FIG. 5.

The YMYL sequence from NiV M can restore budding of late-domain mutant EBOV VP40. (a) Illustration of the chimeric EBOV VP40 constructs. WT, wild type. (b and c) 293T cells were transfected with the indicated plasmids for 48 hours, and the budding assay was performed as described in the legend to Fig. 1. (d) The indicated VLPs were treated with TPCK-trypsin (0.1 mg/ml) in the presence or absence of Triton X-100 as described for Fig. 1. (e) Transmission electron micrographs of budded VP40 VLPs stained with 1% phosphotungstic acid, pH 7.0. Panels: i, VP40 wild-type; ii, VP40 rPT/PY; iii, VP40 rYMYL; iv, VP40 rYMYL with flanking sequences.