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Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly

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Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly

Nicolás Cifuentes-Muñoz et al. J Virol.

Abstract

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

Keywords: Hendra; Rab11; endocytic trafficking; fusion; matrix; virus assembly.

Figures

FIG 1
FIG 1
HeV M protein aids in proper assembly of F into VLPs. (A) 293T cells were transfected to produce the HeV M and F proteins either separately or together. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (B) 293T cells were transfected to produce the HeV F and M proteins either separately or together. VLPs were purified by centrifugation through sucrose cushions, adsorbed to carbon-coated grids, and visualized by transmission electron microscopy after negative staining. The white arrowhead indicates the spike layer present on M+F VLPs. Bars = 100 nm.
FIG 2
FIG 2
Coordination of HeV M and F proteins during VLP assembly. (A) 293T cells were transfected to produce the HeV F and M proteins either separately or together. VLPs were purified by centrifugation through sucrose cushions and then loaded onto the tops of 5% to 45% continuous sucrose gradients. After centrifugation, fractions were collected, and the viral proteins in each fraction were detected by immunoblotting. (B) Vero cells (upper panels) or 293T cells (lower panels) on glass coverslips were transfected to produce the HeV F and M proteins together. Cells were bound with an F-specific antibody, fixed, permeabilized, bound with an M-specific antibody, and then visualized by fluorescence microscopy. (C) 293T cells were transfected to produce the HeV F and M proteins either separately or together. Proteins synthesized in the transfected cells were metabolically labeled, cells were lysed, and immunoprecipitation was carried out using an anti-HeV F antibody or an anti-Myc-tag antibody (to detect the Myc-tagged M protein), as indicated. Proteins were detected using a phosphorimager.
FIG 3
FIG 3
Removal of the HeV F cytoplasmic tail impairs VLP assembly. (A) Schematic illustration of the HeV F protein and its cytoplasmic tail truncations. (B) Vero cells were transfected to produce the indicated HeV F protein variants. Cells were metabolically labeled, surface proteins were biotinylated, cells were lysed, and immunoprecipitation was carried out using an anti-HeV F antibody. Proteins were detected using a phosphorimager. (C) Vero cells were transfected to produce the HeV F and G proteins, and syncytium formation (black arrowheads) was observed after 18 h posttransfection. (D) 293T cells were transfected to produce the HeV M protein together with the indicated HeV F protein variants. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (E) 293T cells were transfected to produce the F ΔE519 protein alone or the F ΔE519 protein together with the M protein, as indicated. VLPs were purified by centrifugation through sucrose cushions, adsorbed to carbon-coated grids, and visualized by transmission electron microscopy after negative staining. Bars = 100 nm.
FIG 4
FIG 4
Endocytic trafficking of the HeV F protein is required for proper assembly with M. (A) 293T cells were transfected to produce the HeV M protein together with the HeV F protein or the HeV F S490A protein in the presence of the cathepsin inhibitor E-64d, as indicated. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (B) 293T cells were transfected to produce the HeV F protein alone or the HeV F protein together with the M protein, as indicated, in the presence of the cathepsin inhibitor E-64d. VLPs were purified by centrifugation through sucrose cushions and loaded onto the tops of 5% to 45% continuous sucrose gradients. After centrifugation, fractions were collected, and the viral proteins in each fraction were detected by immunoblotting.
FIG 5
FIG 5
Severe defects to F protein endocytic trafficking correlate with poor VLP assembly. (A) Schematic illustrating the locations of HeV F protein mutations that affect its endocytic trafficking. (B) Table summarizing the defective phenotype of each F mutant relative to the wt phenotype. +++, wt level; +, less than 25% of WT level; −, not detectable. (C) 293T cells were transfected to produce the HeV M protein together with the indicated HeV F protein variants. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (D) The F:M ratios in the VLP fractions were calculated as follows: F:M ratio = (F0 signal plus F1 signal)/M signal. Error bars indicate standard deviations (n = 3). *, P < 0.05.
FIG 6
FIG 6
Severe defects in F protein endocytic trafficking correlate with poor VLP assembly under conditions of limiting M expression. (A) 293T-PSU cells were transfected to produce the HeV M protein together with the indicated HeV F protein variants. The M plasmid amount was reduced to 50 ng per dish (from the usual 200 ng per dish) to achieve conditions of limiting M expression. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (B) 293T-KY cells were transfected to produce the HeV M protein together with the indicated HeV F protein variants. Viral proteins from cell lysates and from purified VLP fractions were detected by immunoblotting. (C) The relative efficiency of VLP production was calculated either based on F protein content (as the amount of F0 plus F1 detected in VLPs divided by the amount detected in cell lysates, normalized to the value obtained with the wt F protein) or based on M protein content (as the amount of M detected in VLPs divided by the amount detected in cell lysates, normalized to the value obtained with the wt F protein) (n = 6). **, P < 0.01; ***, P < 0.001. (D) 293T-KY cells were transfected to produce the indicated HeV F protein variants. Surface-exposed proteins were biotinylated, cells were lysed, the biotinylated fraction was recovered, and the F protein was detected by immunoblotting. The amount of F signal was plotted, normalized to the value obtained with the wt F protein (n = 3). VLP production values obtained from panel C were plotted alongside the cell surface expression values.
FIG 7
FIG 7
Rab11 recycling endosomes play a role in M-VLP assembly. Vero cells (A) or 293T cells (B) on glass coverslips were transfected to produce the HeV M protein, and subcellular localizations of M protein (red) and endogenous Rab11 (green) were visualized by immunofluorescence microscopy. (C) 293T cells were transfected to produce the HeV M protein together with GFP-Rab11 or dominant negative (DN) GFP-Rab11, as indicated. (D) The relative efficiency of VLP production was calculated as the amount of M detected in VLPs divided by the amount detected in cell lysates, normalized to the value obtained in the absence of Rab11 expression.
FIG 8
FIG 8
Rab11 recycling endosomes play a role in F-VLP assembly. Vero cells (A) or 293T cells (B) on glass coverslips were transfected to produce the HeV F protein, and subcellular localizations of F protein (red) and endogenous Rab11 (green) were visualized by immunofluorescence microscopy. (C) 293T cells were transfected to produce the HeV F protein together with GFP-Rab11 or dominant negative GFP-Rab11, as indicated. (D) The relative efficiency of VLP production was calculated as the amount of F0 plus F1 detected in VLPs divided by the amount detected in cell lysates, normalized to the value obtained in the absence of Rab11 expression.

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