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Proteomic analysis of RABV requires purified virions with high purity and an intact structure of typical bullet-shaped morphology. Supernatants of large-scale cultures of CVS-11 with a titer of 108.05 TCID50/mL, were harvested and the virus was purified by ultracentrifugation, resulting in five visible fractions in the iodixanol density gradient (F0-F4) as shown in Fig. 1A. Observation by TEM showed that most particles are in well-defined bullet-shaped with a length of 120-180 nm in the F3 band (20%-25% iodixanol). The truncated or crescent defective interfering (DI) particals with length of 50-70 nm can also be found in the F1 (10%-15% iodixanol) and F2 (15%-20% iodixanol) bands. No virus particles were apparent in the F0 and F4 bands (Supplementary Figure S1). Following further sedimentation through a 15%-25% iodixanol density gradient, the purity and integrity of the virions of three independent purifications were checked by electron microscopy prior to the following proteomic analysis. As shown in Fig. 1B, the purified virus particles were free of cellular debris or vesicles with most having intact, typical bullet shapes of length 120-180 nm. Small numbers of ruptured and DI particles were observed (Fig. 1B). Higher magnification highlighted the characteristic bullet-shaped morphology covered by numerous spikes (Fig. 1C). The purity of the virion preparations was estimated by counting the proportion of virus particles among total particles in images of the three sets of purifications. Of a total 1200 counts, 95.6% were RABV, consisting of 87% intact virions and 8.6% of damaged or DI particles.
Figure 1. Purification and electron microscopy (EM) of RABV particles. A Ultracentrifugation of RABV particles in a 10%-30% discontinuous iodixanol density gradient. B The images of purified RABV virions by EM (× 10, 000) show multiple intact bullet-shaped virions (black arrow) as well as a few damaged (black arrow head) and DI particles (white arrow head). C High magnification (× 40, 000) of intact bullet-shaped RABV virions, highlight the G protein spikes covering the viral surface. D A typical cryo-EM image of intact bullet-shaped RABV virions at × 96, 000 clearly showing four distinct layers from the outside to the inside: glycoprotein spike, viral envelope (lipid bilayer), M protein helix and RNP complex.
Cryo-electron microscopy (cryo-EM) has become a robust imaging tool for determining the ultrastructure of viruses at near-atomic resolution (Yuan et al.2018). Images clearly showed four distinct electron-dense layers (Fig. 1D), in which the outermost layer, on the viral surface, consisted of spikes (G protein molecules) over the entire surface apart the planar end. The lipid bilayer of the envelope covering the entire capsid formed the second layer. The third layer consisted of helical structures (M protein molecules), similar to that of vesicular stomatitis virus (VSV) (Ge et al.2010). However, the EM images showed that both G and M layers of the rabies virions were absent from the planar ends, the result is consistent with that of previous publication (Guichard et al.2011). The innermost layer was super-helical structure known to consist of the ribonucleoprotein (RNP) complex VSV (Ge et al.2010) and RABV (Luo et al.2007; Riedel et al.2019).
In order to understand the proteomic characteristics of rabies virions prepared from infected animals, attempts were made to purify the virus from CVS-11 infected mouse brain tissues. Unfortunately, the experiment with various iodixanol density gradients and centrifugation programs failed to remove all brain tissue debris from the virion preparations. Therefore the virus grown on N2a cell lines was eventually used for the proteomic analysis.
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SDS-PAGE of purified virions revealed six bands, including two forms of differentially glycosylated viral glycoprotein (GI and GII) (Fig. 2A). Following deglycosylation only one band (G0) was seen. Both glycosylated and deglycosylated G proteins were further identified by Western blotting (Fig. 2B). In addition, analysis also revealed some fainter bands that may represent a low abundance of cellular proteins (Fig. 2A). These results showed that the deglycosylation of proteins in purified virions was complete and that the virion proteins could be used for proteomic analysis.
Figure 2. Electrophoresis and Western blot analysis of RABV virion proteins. A SDS-PAGE of the proteins of purified virions (lane 1) and following deglycosylation (lane 2). Eight µg purified virions was loaded in each lane; B Western blot analysis of the viral G protein with its specific polyclonal antibody: two forms of G protein, GI and GII, were detected in purified virions (lane 1), while only one form, G0, was detected following deglycosylation (lane 2).
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Nano LC-MS/MS was utilized to identify the protein composition of purified rabies virions, resulting in identification of 54 high-confidence proteins. These were considered to be incorporated into mature virions based on the following criteria: (1) the proteins were identified in each of three independently purified virion preparations; (2) the abundance of target proteins exceeded 106; (3) each target protein had at least 2 unique peptides. Proteins identified in low abundance or as being unreproducible were likely randomly loaded contaminants or sticky proteins, and were therefore excluded from the viral proteomic composition. Table 1 lists all high-confidence proteins ranked according to their average abundance values. The results are consistent with SDS-PAGE analysis, both showing that the five viral structural proteins were most abundant.
Protein ID Protein name Description Unique peptidesa Sequence coverage (%)b Abundancec Mass(kDa) Subcellular locationd Reported in other virusese O92284 G Glycoprotein 37 49 1.75E+10 58.86 Virion Q8JXF6 N Nucleoprotein 40 80.7 1.48E+10 50.73 Virion P22363 P Phosphoprotein 32 84.2 1.13E+10 33.62 Virion P25223 M Matrix protein 8 52.5 7.75E+09 23.13 Virion D8VEC2 L Large structural protein 111 61.5 3.94E+09 242 Virion P40240 CD9 CD9 antigen 7 26.5 1.72E+09 25.26 Membrane IAV1, HAV, MEV, HIV1, ASFV P63168 DLC8 Dynein light chain 1 2 57.3 1.09E+09 10.37 Nucleus, mitochondria, cytoskeleton P63017 HSC70 Heat shock cognate 71 kDa protein 28 55.9 5.83E+08 70.87 Membrane, nucleus, cytoplasm RSV, HIV1, HIV2, VSV, RVFV, HSV, ASFV, JUNV P17742 CyPA Cyclophilin A 9 68.3 5.65E+08 17.97 Cytoplasm IAV1, MEV, HIV1, HIV2, HIV3, HSV, KSHV P63001 RAC1 RAS-related C3 botulinum 6 36.5 3.51E+08 23.43 Membrane, cytoplasm RVFV, HIV2 P18760 Cofilin-1 Cofilin-1 14 58.1 3.27E+08 24.58 Cytoskeleton IAV1, RSV, HIV1, HIV2, HSV P35762 CD81 CD81 antigen C 4 30.9 2.99E+08 25.81 Membrane IAV1, HCV, MEV, HIV1, HIV2, VV P61205 ARF3 ADP-ribosylation factor 3 4 57.5 2.93E+08 20.60 Golgi HSV P0CG50 Ubc Polyubiquitin-C 7 73.6 2.41E+08 82.55 Nucleus, cytoplasm IAV2, RSV, HIV1, VSV, JUNV O08992 Syntenin-1 Syntenin-1 8 40.5 1.77E+08 32.38 Membrane, cytoplasm, cytoskeleton, nucleus, ER, junction HIV2 Q9WVE8 PACSIN 2 Protein kinase C and casein kinase substrate in neurons protein 2 13 27 1.45E+08 55.83 Cytosol, endosome, nucleus, caveola P10852 Slc3a2 4F2 cell-surface antigen heavy chain 19 39.7 1.03E+08 58.34 Membrane RSV, HIV1, VSV O35566 CD151 CD151 antigen 5 17 8.78E+07 28.25 Membrane RVFV P41731 CD63 CD63 antigen 3 10 8.72E+07 26.78 Membrane HCV Q9R0P5 Destrin Destrin 6 41.2 8.62E+07 18.52 Cytoskeleton IAV1 P60766 CDC42 Cell division control protein 42 homolog 6 42.4 8.24E+07 21.26 Membrane, cytoskeleton, cytoplasm IAV2, HIV1, SARS Q9D8B3 CHMP4B Charged multivesicular body protein 4b 7 41.1 6.07E+07 24.94 Late endosome, cytosol HAV P63037 HSP40 DnaJ homolog subfamily A member 1 11 36.5 5.91E+07 44.87 Membrane, nucleus, ER, mitochondria, cytoplasm HIV2 P16858 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 9 30.6 5.73E+07 38.65 Cytoskeleton, cytosol, nucleus IAV1, RSV, HIV1, HIV2, HIV3, RVFV, IBV, ASFV, KSHV P63242 EIF5a Eukaryotic translation initiation factor 5A-1 6 45.5 5.71E+07 16.83 Nucleus, ER KSHV Q4VAE6 RhoA Ras family member A 5 28.5 5.67E+07 21.80 Nucleus, ER, cytoplasm IBV O54946 HSJ-2 DnaJ homolog subfamily B member 6 6 21.9 5.16E+07 39.81 Nucleus P63024 VAMP3 Vesicle-associated membrane protein 3 2 32 4.84E+07 11.48 Membrane Q9WU78 ALIX Programmed cell death 6-interacting protein 30 38.9 4.84E+07 96.31 Cytoskeleton, cytosol HAV, HIV1, VSV, HSV, JUNV P63101 14-3-3 ζ/θ 14-3-3 protein zeta/delta 6 37.6 4.66E+07 27.77 Cytoskeleton HIV1, HSV, KSHV, SARS Q62167 DDX3X ATP-dependent RNA helicase DDX3X 5 41.2 4.13E+07 73.10 Mitochondria, nucleus HSV, JUNV, SARS P99024 Tubulin β-5 Tubulin beta-5 chain 4 35.8 4.05E+07 49.67 Cytoplasm, cytoskeleton, microtubules IAV1, RSV, HIV1, VSV, ASFV P11499 HSP90β Heat shock protein HSP 90-beta 15 34.4 3.94E+07 83.28 Membrane, cytoplasm, nucleus RSV, HAV, HIV1, VSV, RVFV, IBV, KSHV, SARS Q91ZR2 SNX Sorting nexin-18 10 20 3.76E+07 67.79 Membrane Q61187 TSG101 Tumor susceptibility gene 101 protein 7 19.2 3.40E+07 44.12 Endosome, nucleus HIV1, VSV, JUNV P17182 ENO1 Alpha-enolase 9 24 3.17E+07 47.14 Membrane, cytoplasm IAV1, HAV, MEV, HIV1, HIV2, VSV, IBV, ASFV, KSHV Q9DB34 CHMP2A Charged multivesicular body protein 2a 5 18 3.09E+07 25.13 Cytoplasm, late endosomes Q9Z127 SLC7 Large neutral amino acids transporter small subunit 1 5 8.8 2.94E+07 55.87 Membrane, cytosol P35278 Rab5C Ras-related protein Rab-5C 4 19.7 2.91E+07 25.35 Membrane, endosomes HAV, HIV1, HIV2, ASFV, HSV Q3UFR4 SLC1 Amino acid transporter 8 18.9 2.60E+07 58.36 Membrane Q99J93 IFITM Interferon-induced transmembrane protein 2 2 23.6 2.30E+07 15.74 Membrane Q9D1C8 VPS28 Vacuolar protein sorting-associated protein 28 homolog 7 36.7 2.19E+07 25.45 Endosomes HIV2 P51150 RAB7A Ras-related protein Rab-7a 6 32.9 1.88E+07 23.49 Late endosomes HAV, HIV1, RVFV, HSV, KSHV, JUNV P06837 GAP43 Neuromodulin 7 49.8 1.79E+07 23.63 Membrane, synapses P26040 Ezrin Ezrin 8 18.3 1.77E+07 69.41 Cytoskeleton, cytosol IAV2, HIV1 P60335 PCBP1 Poly(rC)-binding protein 1 4 18.5 1.64E+07 37.5 Nucleus, cytoplasm HIV1, HIV2, SARS P16045 Galectin-1 Galectin-1 3 28.1 1.61E+07 14.87 Cell surface, extracellular matrix HIV2 P62331 ARF6 ADP-ribosylation factor 6 3 21.1 1.11E+07 20.08 Cytoplasm, cytosol, early endosomes Q91YD9 nWASP Neural Wiskott-Aldrich syndrome protein 5 12.6 9.01E+06 54.27 Cytoplasm, cytoskeleton, nucleus P61089 Ube2 Ubiquitin-conjugating enzyme E2 N 5 33.6 6.31E+06 17.14 Cytoplasm, nucleus B2RRX1 β-actin Beta-actin 3 54.1 4.96E+06 41.74 Membrane, cytoskeleton, cytosol IAV1, HIV1, HIV2, VSV, RVFV, IBV, HSV, KSHV Q8R0J7 VPS37B Vacuolar protein sorting-associated protein 37B 4 17.9 4.30E+06 31.06 Late endosomes, cytoplasm P46467 VPS4B Vacuolar protein sorting-associated protein 4B 7 18.2 3.62E+06 49.42 Late endosomes HAV P68040 RACK1 Receptor of activated protein C kinase 1 4 11.7 2.58E+06 35.08 Membrane, cytoplasm, nucleus aThe number of unique peptides correspond to the maximal values among the three biological replications.
bThe percentages of sequence coverage based on peptides with unique sequences.
cAverage abundance expressed by iBAQ calculated from three separate determinations.
dSubcellular location was investigated using the Uniprot database. Endoplasmic reticulum, ER.
eVirus names: influenza A virus (Shaw et al.2008 for IAV1,Mindaye et al. 2017 for IAV2); HAV: hepatitis A virus (McKnight et al.2017 ); MEV: measles virus (Sviben et al.2018 ); HIV: human immunodeficiency virus (Linde et al.2013 for HIV1,Chertova et al. 2006 for HIV2,Saphire et al. 2006 for HIV3); ASFV: African swine fever virus (Alejo et al.2018 ); RSV: respiratory syncytial virus (Radhakrishnan et al.2010 )); VSV: vesicular stomatitis virus (Moerdyk-Schauwecker et al.2009 ); RVFV: rift Valley fever virus (Nuss et al.2014 ); HSV: herpes simplex virus type 1 (Stegen et al.2013 ); KSHV: Kaposi's sarcoma-associated herpesvirus (Zhu et al.2005 ); HCV: hepatitis C virus (Lussignol et al.2016 ); VV: vaccinia virus (Krauss et al.2002 ); IBV: infection bronchitis virus (Kong et al.2010 ); JUNV: Junin virus (Ziegler et al.2018 ); SARS: severe acute respiratory syndrome (Neuman et al.2008 ).Table 1. High-confidence proteins identified in purified RABV virions by LC–MS/MS.
The 49 cellular proteins had significant abundance values ranging from 1.72E+09 to 2.58E+06, with sequence coverages of between 73.6% and 8.8%. Table 1 also lists their subcellular locations and incorporation into other virions as reported elsewhere. According to the Uniprot Knowledge database, many of the proteins were localized at the cell membrane, cytoplasm and actin cytoskeleton. It is worth noting that more than half of these host proteins (34/49) were also found in the other viruses within 11 viral families (Table 1). Of them, CD9, CD81, cofilin-1, cyclophilin A, GAPDH, enolase, HSC70, HSP90β and RAB5C were the most frequently identified in the virions of different viruses, indicating that they were widely recruited by these viruses to benefit their replicative cycles (Table 1). For example, the presence of cyclophilin A in the virions aids capsid stabilization in both influenza virus and HIV by interacting with capsid protein in the early stage of the viral replication (Liu et al. 2009, 2016). As reviewed in a recent publication, heat shock protein 90 is a crucial host factor required by many viruses for multiple phases of their life cycle (Wang et al.2017). These suggest that many viruses, especially enveloped ones, might utilize cellular proteins to complete their replicative cycle.
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To justify the claim of incorporation, 3 viral proteins and 11 host proteins from Table 1 of different abundances were analyzed by Western blotting and immunogold labeling. A protease protection assay was performed using ProK to degrade any proteins on the surface of the virions, while internal proteins were protected by the lipid envelope. Absence of the viral glycoprotein and the presence of the viral nucleoprotein (N) and matrix protein (M) verified that the ProK digestion was 100% efficient (Fig. 3A). Immunoblotting analysis revealed that HSC70, cofilin, CHMP4B, HSP40, ALIX, TSG101, CHMP2A, VPS37 and VPS4B were all present in the ProK-treated virions, indicating their locations within the virion (Fig. 3A). Extracts from uninfected and infected N2a cells were included as controls to confirm the reactivity of the antibodies and size of the proteins. CD9, however, was completely absent from ProK-treated virions while β-actin was reduced by about 50%, indicating that CD9 was present only on the virion surface, while β-actin was located both inside and outside (Fig. 3A). Immunogold labeling further confirmed the incorporation of these host proteins into the virion surface. As shown in Fig. 3B, gold particle labelling identified CD9 and β-actin as well as the viral G protein on the surface of virus particles. Although it was unrealistic to validate all 49 cellular proteins, confirmation of the incorporation of all 11 proteins demonstrated the high probability of incorporation of all 49 proteins into rabies virions. Clearly, the potential roles of virion-packaged host proteins during RABV infection merit further investigation.
Figure 3. Validation of cellular proteins incorporated into the RABV virions. A Detection of 3 viral proteins and 11 cellular proteins by Western blotting from: mock-infected N2a cells (lane 1); CVS-11-infected N2a cells (lane 2); purified virions (lane 3); and ProK-treated virions (lane 4). B Images of immunogold labeling of purified RABV virions targeting the following proteins: RABV G protein, cellular CD9 and β-actin. IgG, included as a control for unrelated immunogold-labeled antibody, did not show colloidal gold particles on the virions.
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As is well known, many viruses hijack cellular machinery via host-pathogen interactions to function in their replicative cycles (Robinson et al.2018). To better understand the biological significance of the host proteins ending up in mature rabies virions, functional predictions of the 49 virion-packaged cellular proteins were performed according to the Gene Ontology Database. Results showed that they are grouped into 12 functional categories, including viral transport, protein localization, cytoskeleton organization, and transcription (Fig. 4, Supplementary Table S1). Some proteins had multiple functions, and therefore were classified into multiple categories. Worth noting is that 24 of the 49 proteins were associated with viral processes such as entry (Galectin-1, CD9, CD81, CD151, CD63, IFITM2), genome replication (PCBP1, HSC70, ARF3, CyPA, DDX3X, ENO1, IFITM2, Rack1), assembly (HSP90β), budding (ALIX, CHMP4B, CHMP2A, VPS4B, TSG101, VPS37B, VPS28), release (Rab-7A, cofilin-1) and spread (PACSIN2) (Fig. 4 and Supplementary Table S1).
To identify clusters and interactions among the above host proteins, a protein-protein interaction (PPI) network was constructed by Metascape using the BIOGRID6, InWeb_IM7 and OmniPath8 databases (https://metascape.org/). Additionally, the Molecular Complex Detection (MCODE) algorithm was applied to screen densely connected protein groups in the network and to annotate the biological functions of each group. As a result, the 49 cellular proteins were found to form a PPI network with 24 nodes and 25 edges, as shown in Fig. 5, in which three significant modules were identified by the MCODE algorithm to be linked to 9 cell processes (Supplementary Table S2). Enrichment of the MCODE 2-related viral budding process was the most significant with the smallest p-value of 10-21.7, strongly implying the association of the candidate host proteins with viral budding through the endosomal sorting complex required for transport (ESCRT).
Figure 5. PPI map of host proteins. Application of the Molecular Complex Detection (MCODE) algorithm to identify densely connected network components. The 49 cellular proteins correspond to 24 nodes (red, blue, and green) and 25 edges (gray) in PPI network, respectively. Three significant MCODEs are displayed on the map by coloring the corresponding nodes.