Nipah Virus-Like Particle Egress Is Modulated by Cytoskeletal and Vesicular Trafficking Pathways: a Validated Particle Proteomics Analysis

Abstract

Classified as a biosafety level 4 (BSL4) select agent, Nipah virus (NiV) is a deadly henipavirus in the Paramyxoviridae family, with a nearly 75% mortality rate in humans, underscoring its global and animal health importance. Elucidating the process of viral particle production in host cells is imperative both for targeted drug design and viral particle-based vaccine development. However, little is understood concerning the functions of cellular machinery in paramyxoviral and henipaviral assembly and budding. Recent studies showed evidence for the involvement of multiple NiV proteins in viral particle formation, in contrast to the mechanisms understood for several paramyxoviruses as being reliant on the matrix (M) protein alone. Further, the levels and purposes of cellular factor incorporation into viral particles are largely unexplored for the paramyxoviruses. To better understand the involvement of cellular machinery and the major structural viral fusion (F), attachment (G), and matrix (M) proteins, we performed proteomics analyses on virus-like particles (VLPs) produced from several combinations of these NiV proteins. Our findings indicate that NiV VLPs incorporate vesicular trafficking and actin cytoskeletal factors. The involvement of these biological processes was validated by experiments indicating that the perturbation of key factors in these cellular processes substantially modulated viral particle formation. These effects were most impacted for NiV-F-modulated viral particle formation either autonomously or in combination with other NiV proteins, indicating that NiV-F budding relies heavily on these cellular processes. These findings indicate a significant involvement of the NiV fusion protein, vesicular trafficking, and actin cytoskeletal processes in efficient viral particle formation.IMPORTANCE Nipah virus is a zoonotic biosafety level 4 agent with high mortality rates in humans. The genus to which Nipah virus belongs, Henipavirus, includes five officially recognized pathogens; however, over 20 species have been identified in multiple continents within the last several years. As there are still no vaccines or treatments for NiV infection, elucidating its process of viral particle production is imperative both for targeted drug design as well as for particle-based vaccine development. Developments in high-throughput technologies make proteomic analysis of isolated viral particles a highly insightful approach to understanding the life cycle of pathogens such as Nipah virus.

Keywords: Nipah virus; cytoskeleton; endocytosis; host-pathogen interaction; paramyxovirus; proteomics; vesicular trafficking.

FIG 1

Overview of workflow and results from VLP and cellular omics. (A) Human embryonic kidney (HEK293T) cells were transfected with combinations of constructs coding for the Nipah virus fusion (F), attachment (G), and matrix (M) proteins. Media and cells were collected after 48 h. The medium supernatant was used to isolate virus-like particles (VLPs), and then both VLPs and cells were prepared for proteomics analysis. (B) VLPs produced from the listed combinations of viral protein transfection were isolated and negatively stained with 1.5% uranyl acetate. Scale bars = 250 nm. (C) Venn diagram summarizing the number of proteins identified as enriched in each VLP combination and their distribution of overlap. The black circle highlights the significant overlap between combinations including F.

FIG 2

VLP proteomics highlighted by enrichment of cytoskeletal and endosomal trafficking machinery. (A) STRING protein interaction map comparing proteins identified in the FGM VLPs described by Vera-Velasco et al. (43) (blue box) and this study (red boxes). This map identifies which protein clusters have associated factors included in one or both (purple boxes) studies. Among the most enriched groups based on gene ontological analyses of the combined data set are vesicular trafficking (yellow) and the cytoskeleton (light blue), with proteins that belong to both also designated (green). ESCRTs which somewhat overlap both processes were observed in both studies and are in a black circle. Note that the clusters associated with these processes tend to have included proteins identified in both studies analyzed. Importantly, only proteins with at least one interaction were shown on this map; a full list can be found in Table 1. One of the most clearly enriched proteins in VLP proteomics for this study and that by Vera-Velasco et al. (43) was the autophagy adapter protein, sequestosome-1 (SQSTM1, designated by small black star). (B) Using Western blot analysis of VLPs for each combination, sequestosome-1 was used to validate the accuracy of our VLP proteomics, since this protein exhibited an expression pattern similar to the mass spectrometry results. The Western blot is representative of at least three replicates.

FIG 3

Nipah virus budding is significantly modulated upon the inhibition of endocytosis, recycling, and ESCRT function. (A) HEK293T cells were transfected with NiV F alone or with dominant-negative (DN) mutants of the following key vesicular trafficking factors: dynamin (endocytosis), Eps15 (clathrin-mediated endocytosis), VPS4A (ESCRT function), or Rab11 (recycling). Total cell lysates (CL) and virus-like particle (VLP) fractions were prepared 24 h after transfection and used for SDS-PAGE and Western blot analysis, whereas F cell surface expression (CSE) was measured using flow cytometry. (B) Using densitometry to assess the expression of the F protein in VLPs along with corresponding total cell lysate and CSE values, two indices were determined, VLP/CL and VLP/CSE. (C and D) As with panels A and B, a construct encoding G was coexpressed with each DN factor and the following CL, CSE, VLP, and index values elucidated. Similarly, coexpression with these constructs were tested for effects on M expression in cell lysates and VLPs but not at the cell surface due to it being a cytoplasmic protein. (E and F) Representative Western blots (E) and quantification (F) are shown. The results are representative of at least three experiments, with error bars indicating the standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiency indices compared to when they are cotransfected with pcDNA3.1, an empty vector (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 4

Nipah virus budding is significantly affected by actin cytoskeletal manipulation through mutant RhoGTPases. (A and B) HEK293T cells were transfected with NiV F alone or with dominant-negative (DN) or constitutively active (CA) constructs for RhoA, Rac1, and Cdc42. As with Fig. 3, CL, CSE, and VLPs were quantified and both budding indices assessed. SDS-PAGE and Western blot analysis. (C to F) The effects of coexpressing these mutants were assessed for G-driven (C and D) and M-driven (E and F) budding. The results are representative of at least three experiments, with error bars indicating the standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiency indices compared to when they are cotransfected with pcDNA3.1, an empty vector (*, P < 0.05; **, P < 0.01).

FIG 5

Inhibition of Nipah virus fusion protein budding reduces attachment protein incorporation despite the presence of normally budding matrix protein. (A) To assess the effects that manipulation of vesicular trafficking (Fig. 3) and the actin cytoskeleton (Fig. 4) have on more complete budding particles, NiV F, G, and M were cotransfected with or without DN-dynamin, DN-Vps4a, DN-Rab11, or CA-RhoA. The expression of each viral protein in cell lysates, on the cell surface for F and G, and in VLPs was quantified and used to produce budding indices as done previously. (B to D) These values are summarized for F (B), G (C), and M (D). The results are representative of at least three experiments, with error bars indicating the standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiencies for each viral protein compared to FGM coexpression alone (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 6

Loss of increased G incorporation from DN-dynamin expression occurs with expression of F but not G. HEK293T cells were transfected with constructs for NiV G and either F or M with or without the additional expression of DN-dynamin. As with Fig. 3 and 5, VLPs were collected compared with total and surface expression to assess the G budding efficiency. (A and B) Western blots (A) along with densitometric and surface expression (B) were quantified. The results are representative of at least three experiments, with error bars indicating the standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiencies for each viral protein compared to FGM coexpression alone (*, P < 0.05; **, P < 0.01).

FIG 7

Expression of select mutant factors that inhibit F budding does not affect processing but can modulate fusion. (A) HEK293T cells were transfected with an empty vector or with NiV F and G with or without the listed cellular factors. Light microscope images were taken at ×200 magnification. (B) By counting nuclei in fused syncytia, levels of fusion were quantified, with F and G cotransfection alone set to 100%. Levels of F processing as well as F and G CSE levels were quantified from FGM cotransfections. The results are representative of at least three experiments, with error bars indicating standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiencies for each viral protein compared to FGM coexpression alone (*, P < 0.05; **, P < 0.01).