Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells

Purification of MrNvc VLPs

Expression and purification of the MrNvc and its N-terminal deletion mutants (9ΔMrNvc, 19ΔMrNvc, 29ΔMrNvc and 20–29ΔMrNvc) were as described previously by Goh et al. (2011) and Goh et al. (2014). Briefly, E. coli cells harbouring the recombinant plasmids were grown in Luria-Bertani broth (500 ml) containing ampicillin (50 mg/ml) at 220 rpm for overnight. E. coli cultures were induced for recombinant protein expression with IPTG (1 mM) at 37 °C for 5 h. Cells were then pelleted and lysed in lysis buffer (25 mM HEPES, 500 mM NaCl; pH 7.4) by adding phenylmethylsulfonyl fluoride (PMSF, 2 mM), MgCl₂ (4 mM), freshly prepared lysozyme (0.2 mg/ml) and DNase 1 (0.02 mg/ml). After 2 h of incubation at room temperature (RT), the cells were sonicated at 200 Hz, 15 times with 15 s interval. The mixture was centrifuged at 10,000×  g and supernatant was loaded into HisTrap HP columns (1 ml; GE Healthcare, Buckinghamshire, United Kingdom). Washing buffer A (25 mM HEPES, 500 mM NaCl, 50 mM imidazole; pH 7.4) and B (25 mM HEPES, 500 mM NaCl, 200 mM imidazole; pH 7.4) were used to wash the unbound proteins. Elution buffer (25 mM HEPES, 500 mM NaCl, 500 mM imidazole; pH 7.4) was used to elute MrNv recombinant proteins. Eluted capsid proteins were dialysed in HEPES buffer (25 mM HEPES, 150 mM NaCl; pH 7.4) at 4 °C, overnight.

Fluorescence microscopy

NHS-fluorescein (1 mg; Thermo Scientific, Rockford, USA) was dissolved in DMSO (100 µl). Dialysed MrNvc VLPs (100 µg) were incubated with NHS-fluorescein solution (0.01 mg/ml) at 4 °C for 24 h and then dialysed in HEPES buffer for 24 h. Sf9 cells (ATCC^® CRL-1711^™; 8 × 10⁵ cells) were seeded on glass coverslips in a 6-well culture plate with Sf900 II SFM medium (2 ml; GIBCO, Grand Island, NY, USA) for 24 h and the medium in each well was changed with a fresh medium. The fluorescein labelled VLPs (F-MrNvc VLPs) were filtered with a membrane filter (0.4 µm) and the filtered VLPs (25 µg) were added into the culture plates in triplicates. Non-labelled MrNvc VLPs were used as negative controls. The cells were incubated for 16 h at RT to allow maximum nuclear translocation of MrNvc. The cells in each well were then incubated with the Cell Tracker™ Orange CMTMR Dye (0.5 µM; Thermo Fisher Scientific, Massachusetts, USA) in medium (2 ml) for 30 min to stain the cell cytoplasm. The coverslips were then washed with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄; pH 7.4) for six times and the cells were fixed with paraformaldehyde (3.7% in PBS; pH 7.4) for 10 min at RT. One drop of NucBlue Live Ready Probe Reagent (Life Technologies, Carlsbad, CA) was added to the mounting medium (1 ml; 90% glycerol, 20 mM Tris–HCl (pH 8.5) and 100 mM propyl gallate) to stain the cell nucleus. A drop of mounting medium was placed on a glass slide and the coverslip was carefully put on top of it. The edge of the coverslip was sealed with a nail polish and the slides were observed under a fluorescence microscope, LEICA DM2500 (Leica Camera, Solms, Germany) with green (excitation filter BP 515–560; emission filter LP 590), blue (excitation filter BP 450–490; emission filter LP 515) and red (excitation filter BP 590–650; emission filter BP 700/75) filters under 40× objective.

Z-stack images were captured through the use of a confocal fluorescence microscope, Olympus IX81 (Olympus, Tokyo, Japan) equipped with Disk Scanning Unit (DSU) for spinning disk confocal (pinhole diameter 50–300 µm (1 µm step)) under 60× objective.

Sub-cellular fractionation

Fractionation of Sf9 cells was done according to the method described by Guo, Dallmann & Kwang (2003) with slight modifications on the buffers used. Confluent Sf9 cells in flasks (75 cm²) were added with medium (10 ml) containing MrNvc VLPs (1 mg) and incubated for 16 h. The subsequent steps were done at 4 °C. Cells were washed with ice-cold PBS (pH 7.4) for three times. PBS (3 ml; pH 7.4) containing the Protease Inhibitor Cocktail (10 µl; SIGMA Chemical Co., St. Louis, MO, USA) was added before dislodging the cells from the culture flasks with a cell scraper. The cells were harvested by centrifugation at 260× g for 5 min. Hypotonic buffer (20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 0.1 mM Na₂HPO₄, 20 mM NaF, 10% glycerol, 0.1% tween-20, 1 mM DTT, 1 mM PMSF and 1% Protease Inhibitor Cocktail) was used to resuspend the cell pellets. The suspension was incubated for 30 min on ice. The suspension was vortexed for 1 min and incubated on ice for 1 min, and this process was repeated for five times. The cytoplasmic extract of Sf9 cells was retrieved by centrifugation at 11,330× g at 4 °C for 10 min. The remaining pellet was washed with hypotonic buffer and centrifuged with the same condition. The nuclear pellet was resuspended in hypotonic buffer (without tween-20) with the addition of NaCl (420 mM) and glycerol (20%). The suspension was vortexed as above for 5 times. Then, the nuclear lysate was centrifuged at 11,330×  g at 4 °C for 10 min. The supernatant containing nuclear extract was collected for further analysis with SDS-PAGE and Western blotting.

SDS-PAGE and Western blot analysis

Cytoplasmic and nuclear extracts of Sf9 cells incubated with the VLPs were electrophoresed in SDS-polyacrylamide (12%) gels at 16 mA. The gels were stained with the Coomassie Brilliant Blue (CBB) dye. For Western blotting, the proteins on the gels were transferred onto nitrocellulose membranes and blocked with skimmed milk (10%; Anlene, Auckland, New Zealand) in TBS buffer (50 mM Tris–HCl (pH 7.4) and 150 mM NaCl) for 1 h. The membranes were washed three times with TBST (50 mM Tris–HCl (pH 7.4), 150 mM NaCl and 0.1% (v/v) Tween-20) for 5 min each, and then incubated with anti-His antibody (1:5000 dilution with TBS; GE Healthcare, Buckinghamshire, United Kingdom) or rabbit anti-MrNv serum (1:1000 dilution) at 4 °C. After an overnight incubation, the membrane was washed again with TBST for five times, 5 min each and incubated with anti-mouse IgG antibody conjugated to alkaline phosphatase (1:5000 dilutions in TBS; KPL Inc., Maryland, USA) or anti-rabbit IgG (1:5000 dilution with TBS; SIGMA Chemical Co., St. Louis, MO, USA) overnight at 4 °C. After washing five times with TBST, the protein bands were developed by adding 5-bromochloroindolyl-phosphate (3.3 µl/ml) and nitrobluetetrazolium (6.6 µl/ml) in alkaline phosphatase buffer (100 mM Tris–HCl (pH 9.5), 100 mM NaCl and 5 mM MgCl₂).

Live cell imaging

Sf9 cells (8 × 10⁵ cells) were seeded in 6-well culture plates at RT for overnight. Fresh Sf900 II SFM medium (2 ml) containing F-MrNvc VLPs (25 µg/ml) was added into the cells and incubated for 1 h at 4 °C, followed by 30 min incubation at RT to initiate internalisation of the MrNvc VLPs. NucBlue Live Ready Probe Reagent (1 drop) and 3 mM propyl gallate were added to stain the nucleus prior to viewing the uptake of MrNvc VLPs by Sf9 cells across the plasma membrane in real-time with Olympus IX81-DSU equipped with a high-sensitivity cooled CCD (charge-coupled device) camera with a built-in shutter, which allows single DSU image to be obtained in 0.1–0.4 s and avoid photobleaching when no image is acquired. Fluorescence images were visualised by exciting the fluorescence at 493 nm (green for VLPs) and 360 nm (blue for cell nucleus). Static superimposed images were captured every 30 s for 1 h duration and compiled into a video to observe the uptake of VLPs by the cells.

Endocytosis inhibition assay

Five endocytosis inhibitors were used to study the uptake mechanism of MrNvc VLPs into Sf9 cells. The Sf9 cells (8 × 10⁵ cells) were seeded on a glass slide in each well of 6-well culture plates for overnight. The Sf900II SFM medium was removed and the cells were pre-incubated in 2 ml medium for 1 h at RT with: cytochalasin D (2 µM; Calbiochem, CA, USA), NH₄Cl (10 mM; Bio Basic Inc., NY, USA), chlorpromazine (CPZ: 50 µM; Santa Cruz Biotechnology, Texas, USA), methyl-β-cyclodextrin (2 mM; Sigma-Aldrich, MO, USA) and genistein (100 µM; Calbiochem, CA, USA). The cells were then incubated for another 16 h at RT following the addition of F-MrNvc VLPs (25 µg/ml). The coverslips were washed with PBS (pH 7.4) for six times and the cells were fixed with paraformaldehyde (3.7% in PBS; pH 7.4) for 10 min at RT. A drop of mounting medium was placed on a glass slide and the coverslip was gently placed on top of it. The edge of the coverslip was sealed with a nail polish and the slides were observed under the Zeiss LSM5 PASCAL laser scanning microscope (Carl Zeiss, Oberkochen, Germany) excited with 488 nm laser (pinhole diameter 160 µm) under 63× objective.

Cell viability assay

The viability of Sf9 cells incubated in different endosomal inhibitors were analysed with 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Sf9 cells (100 µl; 2 × 10⁵ cells/ml) were seeded on 96-well culture plates and incubated for 16 h at RT in a medium (100 µl) containing cytochalasin D (2 µM), NH₄Cl (10 mM), chlorpromazine (50 µM), methyl-β-cyclodextrin (2 mM) and genistein (100 µM). Sf9 cells without the inhibitors served as controls. The MTT reagent (10 µl; 5 mg/ml in PBS (pH 7.4)) was added in each well and incubated at RT. After 2 h, the medium was removed and MTT solubilisation solution (100 µl; 1:9 part of 10% SDS:DMSO) was added. The plate was incubated for 15 min in dark and the absorbance at 570 nm was measured with the µQuant™ ELISA plate reader (Bio Tek Instruments, Winooski, USA).

The percentage of viable Sf9 cells was measured as (100 − ((A − B)/A × 100)), where A is the absorbance of the control cells and B is the absorbance of treated cells.

MrNvc VLPs internalise Sf9 cells

Fluorescence microscopy was used to examine the delivery and localisation of fluorescein-labelled MrNvc VLPs (F-MrNvc VLPs) in Sf9 cells. The VLPs labelled with NHS-fluorescein at the lysine residues of MrNvc were added to Sf9 cells and incubated at room temperature (Fig. 1). To observe the dispersion of F-MrNvc VLPs in the Sf9 cells, the NucBlue Live Ready Probe Reagent and Cell Tracker Orange were used to stain the nuclear and cytosolic boundaries, respectively. No intense green fluorescence was detected from the normal Sf9 cells (Fig. 1A) and in the Sf9 cells incubated with non-labelled MrNvc VLPs (Fig. 1B). After 16 h of incubation with the F-MrNvc VLPs, green fluorescence accumulated within the Sf9 cells (Fig. 1C), indicating the internalisation of VLPs into the cells. From the merged images, MrNvc VLPs were clearly located at the cytoplasm of Sf9 cells. A close-up examination on the cells showed intense fluorescence spots of small granular appearance localised throughout the cell cytoplasm (Fig. 1D). Interestingly, the fluorescence spots tended to cluster at one side of the cells resembling cap-like structures, which have also been reported by Liu et al. (2005) when SSN-1 cells were infected with dragon grouper nervous necrosis virus (DGNNV), but not the VLPs.

MrNvc distributes in Sf9 cell cytoplasm and nucleus

Sub-cellular fractionation was performed to verify the presence of MrNvc in Sf9 cells. The cells were incubated with MrNvc VLPs at 0, 4, 8, 12 and 16 h. At each time point, the cells were disrupted by hypotonic buffer (HB) to fractionate the nuclear and cytoplasmic components. MrNvc was detected by the rabbit anti-MrNv serum (Fig. 2A(i)) and anti-His antibody (Fig. 2A(ii)). In the cytoplasmic and nuclear fractions of Sf9 cells incubated with MrNvc VLPs, a distinct band corresponding to the MrNvc with a molecular mass ∼46 kDa was detected after 4 h post-incubation. After 8 h of incubation, the MrNvc was detected in the cell nucleus. In addition, a smaller protein band of ∼44 kDa was detected by the serum and anti-His antibody indicating it is an N-terminal degraded product of MrNvc. Goh et al. (2014) and Yong et al. (2015a) also reported that the MrNvc VLPs produced in E. coli contained the N-terminal degraded product. The Sf9 cells incubated with MrNvc VLPs for 16 h were viewed under a fluorescence microscope and 20 Z-stack images were captured. The orthogonal view of the cells was obtained by using the ImageJ software. From the view of XY, YZ and XZ axes, it was confirmed that the MrNvc VLPs, as indicated by the yellow line interceptions, were localised in the cell nucleus (Fig. 2B) and cytoplasm (Fig. 2C).

Effect of endosomal inhibitors on the entry of MrNvc VLPs into Sf9 cells

NH₄Cl (10 mM), cytochalasin D (2 µM), methyl-β-cyclodextrin (2 mM), CPZ (50 µM) and genistein (100 µM) were used to study the entry mechanism of MrNvc VLPs in Sf9 cells. Sf9 cells were pre-incubated with the inhibitors, followed by the addition of F-MrNvc VLPs. After 16 h, the cells were observed under a fluorescence microscope. The amount of green granular appearance in Sf9 cells decreased drastically in the presence of NH₄Cl, CPZ, methyl-β-cyclodextrin and genistein (Fig. 3A(iv–vii)). These results suggest that NH₄Cl, methyl-β-cyclodextrin, CPZ and genistein inhibited MrNvc VLPs entry at the early event of endosomal pathway in Sf9 cells. On the other hand, cytochalasin D did not inhibit the VLPs entry (Fig. 3A(iii)).

MTT assay was performed to assess Sf9 cell viability in the presence of NH₄Cl, cytochalasin D, CPZ, methyl-β-cyclodextrin and genistein. All treated cells have viability percentage over 80% (Fig. 3B), indicating the treatments were not toxic to the cells.

The trafficking mechanism of MrNvc VLPs into Sf9 cells examined with live cell imaging system

The internalisation process of MrNvc VLPs into Sf9 cells was examined using a live cell imaging system. After 1 h pre-incubation at 4 °C, the Sf9 cells were incubated at RT for 30 min. Time lapse video was captured for 1 h in 30 s interval (Video S1). As demonstrated in Fig. 4A, VLPs attached on the cell surface were brought near to a membrane pit, which then accumulated into the middle of the pit (Fig. 4B). The VLPs were enclosed in the endosomes which move freely in the cytosol (Fig. 4C), until the shape became disproportionate (Fig. 4D). The endosome disappeared completely after a few minutes (Fig. 4E as annotated by the red arrow), in which the MrNvc is believed to be released into the cytosol. Endosome formation and endosomal escape are summarised in a schematic diagram (Fig. 4F).

Incubation of Sf9 cells with the N-terminal deletion mutants of MrNvc

The N-terminal region of MrNvc is highly rich in positively charged residues (Goh et al., 2014). The roles of this region in cell entry and internalisation into nucleus were studied by four N-terminally deleted mutants (Fig. 5A); namely 9ΔMrNvc, 19ΔMrNvc, 29ΔMrNvc and 20–29ΔMrNvc. The Sf9 cells incubated with the four deletion mutants and the full length MrNvc were subjected to sub-cellular fractionation (Fig. 5B) and viewed under a fluorescence microscope with two labelling signals (Fig. 5C). Figure 5B shows that the full length MrNvc and the four mutants were detected in the cell cytoplasm by Western blotting. The green signal observed under fluorescence microscopy revealed that the fluorescein-labelled VLPs of all mutants and the full length capsid protein appear as green granules in the cell cytoplasm. This study confirmed the Western blot results demonstrating that the N-terminal region of the MrNvc does not play a role in the entry of VLPs into Sf9 cells, and that the receptor binding site is located somewhere after amino acid 29 of MrNvc. Deletion of amino acids 20–29 (20–29ΔMrNvc) significantly decreased the internalisation of MrNvc into nucleus (Fig. 5B), as no trace of 20–29ΔMrNvc can be detected in the nuclear extract (analysed with the Quantity One Software), demonstrating the importance of these positively charged residues in nuclear localisation.