Materials and reagents

Note: The materials used in this study can be obtained from the corresponding author, Eiji Morita, upon reasonable request.

Biological materials

1. HeLa cells (ATCC, catalog number: CRM-CCL-2)

Note: HeLa cells, passaged at least 50 times in our laboratory, were used. To prevent mycoplasma contamination, cells were stained with Hoechst to check for intracellular DNA, which indicates contamination. Additionally, cells were periodically treated with BM-Cyclin (Roche, catalog number: 10799050001) to remove mycoplasma contamination.

2. Plasmid Str-KDEL_IL-2ss-HiBiT-SBP-GFP1-10 (V206K)

Notes:

1. This plasmid was constructed by replacing the TNF-SBP-EGFP sequence of Str-KDEL_TNF-SBP-EGFP with an in-frame tandem sequence of interleukin-2 signal peptide, HiBiT tag, SBP, and GFP1-10 (V206K).

2. Str-KDEL_TNF-SBP-EGFP is one of the original RUSH constructs reported by Boncompain et al. [7] and is available from Addgene (#65278). RUSH constructs are mammalian expression constructs that tandemly encode a hook protein (Str-KDEL in this case) and a reporter protein [GOI, interleukin-2 signal peptide-HiBiT-SBP-GFP1-10 (V206K) in this case] linked via an internal ribosome entry site sequence. We selected Str-KDEL as the hook protein based on our experimental requirements (see General note 1).

3. The interleukin-2 signal peptide and HiBiT tag sequences were introduced via overlap polymerase chain reaction (PCR). The interleukin-2 signal peptide and SBP sequence was amplified using Ii-Str_ssSBP-EGFP (Addgene #65277) as a template, and the GFP1-10 (V206K) sequence was amplified using ZipGFP1-10_TEV (Addgene #81242) as a template (see General notes 2 and 3). These fragments were then joined using overlap PCR. The resulting DNA fragment was inserted into the AscI/PacI site of Str-KDEL_TNF-SBP-EGFP (see General note 4).

3. Plasmid pCAG-JEprME-S275-GS-GFP11-GS-S276

Notes:

1. This plasmid was constructed by inserting a sequence encoding a GFP11-containing peptide into the E gene of pCAG-prME.

2. The pCAG-prME plasmid, provided by Dr. Ryosuke Suzuki, was generated in a previous study [12]. This plasmid contains the carboxyl-terminal 22 residues of the JEV Nakayama strain C protein (corresponding to the prM protein signal sequence), the full-length prM protein, and the full-length E protein. For detailed information on the pCAG-prME sequence, please contact Dr. Ryosuke Suzuki (National Institute of Infectious Diseases, Japan).

3. A GFP11 peptide sequence, flanked by GS linker sequences at both ends, was inserted in-frame between amino acid residues S275 and S276 of the JEV E protein. The permissive insertion site within the JEV E protein was previously determined via a Flag-tag insertion screening study [10]. For the full-length amino acid sequence of the E protein, refer to Saga et al. [10] (see General notes 5 and 6).

4. Overlap PCR was performed using pCAG-prME as a template with primers containing this sequence, and the GFP11 peptide coding sequence was inserted into the E gene. The resulting DNA fragment was then inserted into the KpnI/XhoI sites of pCAG-MCS2, a pCAGGS [13] derived plasmid vector containing additional multiple cloning sites (see General note 7).

Reagents

1. DMEM (4.5 g/L glucose) without L-Gln, sodium pyruvate, and phenol red, liquid (Nacalai, catalog number: 08489-45)

2. Fetal bovine serum (FBS) (Thermo, catalog number: 10270-106)

3. 100 mM sodium pyruvate solution (100×) (Nacalai, catalog number: 06977-34)

4. 200 mM L-Glutamine stock solution (Nacalai, catalog number: 16948-04)

5. Phosphate-buffered saline without calcium and magnesium (PBS) (Nacalai, catalog number: 14249-24)

6. Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015)

7. OptiMEM (Thermo, catalog number: 31985062)

8. 100 mM biotin stock solution (IBA GmBH, catalog number: 6-6325-001)

9. Sodium hydroxide (Nacalai, catalog number: 31511-05)

10. Hydrochloric acid (Nacalai, catalog number: 18320-15)

11. Paraformaldehyde (Nacalai, catalog number: 26126-25)

12. Triton X-100 (Nacalai, catalog number: 35501-02)

13. Anti-GM130 antibody (BD Biosciences, catalog number: 610822)

14. Anti-mouse IgG antibody, Alexa Fluor 594 conjugated (Jackson ImmunoResearch, catalog number: 115-585-146)

15. Fluoromount-G (Southern Biotechnology Associates, catalog number: 0100-01)

16. MGK-S (Matsunami, catalog number: FX00100)

Solutions

1. Culture media (see Recipes)

2. 1 N NaOH solution (see Recipes)

3. 4% PFA (paraformaldehyde) (see Recipes)

4. 10% TX-100 (w/v) solution (see Recipes)

5. Permeabilizing and blocking solution (see Recipes)

Recipes

1. Culture media

a. 500 mL of DMEM.

b. Add 50 mL of FBS, heat-inactivated by incubation at 56 °C for 45 min.

c. Add 5 mL of 100 mM sodium pyruvate solution (100×).

d. Add 5 mL of 200 mM L-glutamine stock solution.

e. Store at 4 °C.

2. 1 N NaOH solution

a. For 500 mL of 1 N NaOH solution, add 350 mL of Milli-Q water to a glass beaker.

b. Add 20 g of sodium hydroxide drops to Milli-Q water.

c. After completely dissolved, adjust the volume of the solution to 500 mL with water.

d. Transfer the solution to a plastic bottle and store it at room temperature.

3. 4% PFA

a. For 1 L of 4% paraformaldehyde (formaldehyde), add 800 mL of PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Ensure that the solution does not boil.

b. Add 40 g of paraformaldehyde powder to the heated PBS solution.

c. The powder will not immediately dissolve into the solution. Slowly raise the pH by adding 1 N NaOH dropwise using a pipette until the solution clears.

d. Once the paraformaldehyde is dissolved, cool and filter the solution.

e. Adjust the volume of the solution to 1 L with PBS.

f. Recheck the pH and adjust it to approximately 6.9 with small amounts of diluted hydrochloric acid.

Note: To succeed in the downstream process after cell fixation, please pay attention to the pH of the PFA solution.

g. The solution can be aliquoted and frozen or stored at 2–8 °C for up to one month.

4. 10% TX-100 (w/v) solution

a. For 50 mL of 10% TX-100 solution, add 30 mL of Milli-Q water to a 50 mL tube.

b. Add 5 g of Triton X-100 to Milli-Q water and rotate the tube overnight.

c. After the Triton X-100 is dissolved completely, adjust the volume of the solution to 50 mL with Milli-Q water.

d. Store at room temperature.

5. Permeabilizing and blocking solution

a. Dilute 10% TX-100 solution with PBS by 1:100 (v/v). The final TX-100 concentration is 0.1% (w/v).

b. Dilute FBS with PBS by 1:10 (v/v). The final FBS concentration is 10% (v/v)

Laboratory supplies

1. 35 mm glass-bottom dish (Phoenix Science, catalog number: P35G-0-20-C)

2. Humidified incubator (37 °C, 5% CO₂) (PHCbi)

3. 1.5 mL tube (Watson, catalog number: 131-815C)

4. Glass-bottom dish (MatTek Life Science, model: P35G-1.5-14-C)

5. Micro cover glass (Matsunami, thickness: No1S, diameter: 12 mm)

6. White glass, frosted microscope slides (Matsunami, catalog number: S2441)

Equipment

1. Microscope for live imaging based on:

a. EVIDENT IX83 P2ZF inverted fluorescence microscope (Evident, Tokyo, Japan)

b. DP80 CCD digital camera (Evident)

c. IX3-ZDC2 Z-drift compensator (Evident)

d. UPLXAPO 60×/1.35 numerical-aperture oil-immersion objective (Evident, catalog number: UPLXAPO60XO)

e. U-FUNA/U-FBNA/U-FMCHE filter set (Evident)

f. Stage top incubation system (Tokai Hit)

2. Microscope for fixed samples based on:

a. EVIDENT IX83 P2ZF inverted fluorescence microscope (Evident)

b. FV3000 confocal laser scanning microscope (high-speed resonant scanner) (Evident)

c. UPLXAPO 60×/1.35 numerical-aperture oil-immersion objective (Evident, catalog number: UPLXAPO60XO)

d. 5-color laser combiner (Coherent)

Software and datasets

1. cellSens Dimension (Evident)

2. ImageJ/Fiji

Procedure