Detection of HBV C Protein Phosphorylation in the Cell

Materials and Reagents

1. Huh7 hepatoma cells (Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB0403 )
   
2. Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco^®, catalog number: 12800-017 )
   
3. Fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 16000-044 )
   
4. Penicillin/streptomycin (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
5. Plasmid; HBV WT C protein (STSSSS) in plasmid HBV-P-def of pcDNA3 backbone, phosphoacceptor-site mutants (ATAAAA, AAAAAA, SSSSSS, and ASAAAA) in plasmid HBV-P-def in pcDNA3 backbone, pcDNA3-GFP
   
6. Polyethylenimine (Polysciences, catalog number: 23966 )
   
7. Opti-MEM (Life Technologies, Gibco^®, catalog number: 31985-062 )
   
8. OptiMEM (Life Technologies, Gibco^®, catalog number: 31985-070 )
   
9. Dialyzed FBS (Life Technologies, Gibco^®, catalog number: 26400044 )
   
10. 1 mCi orthophosphate [³²P_i] (PerkinElmer Inc., catalog number: NEX053 )
    
11. Polyclonal rabbit anti-HBc antibody (home-made, Jung et al., 2012)
    
12. Protein A/G Plus agarose beads (Calbiochem^®, catalog number: IP05 )
    
13. Tris-HCl (pH 8.5) (Sigma-Aldrich, catalog number: T6066 )
    
14. EDTA (Sigma-Aldrich, catalog number: E5134 )
    
15. Nonidet P-40 (Sigma-Aldrich, catalog number: CA630 )
    
16. NaF (Sigma-Aldrich, catalog number: 201154 )
    
17. β-glycerophosphate (USB, catalog number: 155-56-2 )
    
18. Sodium orthovanadate (Sigma-Aldrich, catalog number: s6508 )
    
19. Protease inhibitors (Calbiochem^®, catalog number: 535142 )
    
20. Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: T6066)
    
21. SDS (Sigma-Aldrich, catalog number: L-3771 )
    
22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    
23. Bromophenol blue (Sigma-Aldrich, catalog number: 114391 )
    
24. Glycerol (USB, catalog number: 16374 )
    
25. Lysis buffer (see Recipes)
    
26. 2x sample buffer (see Recipes)
    

Equipment

1. 10-cm dishes (Corning Incorporated, catalog number: 430167 )
   
2. PVDF membranes (Bio-Rad Laboratories, AbD Serotec^®, catalog number: 162-0177 )
   

Procedure

1. Huh7 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under a humidified atmosphere at 37 °C in 5% CO₂.
   
2. Cells were passaged every three days (passage at 80~90% confluency) and 2 x 10⁶ Huh7 cells were seeded in 10-cm dishes one day before the transfection.
   
3. Next day, Huh7 cells were transfected using polyethylenimine (PEI).
   PEI transfection method:
   
   1. In a sterile tube, total 10 μg of plasmid DNA was mixed with 500 μl Opti-MEM.
      
   2. Add 30 μl of PEI solution (1 μg/1 μl) to DNA-Opti-MEM solution and then vortex immediately.
      
   3. Incubate 15 min at room temperature.
      
   4. Then add PEI/DNA-Opti-MEM mixture to cells.
   For transfections, 10 µg of plasmid encoding HBV STSSSS (WT) or phosphoacceptor-site mutant C proteins in a P-deficient mutant backbone were used. As a transfection control, 1 µg of green fluorescent protein (GFP)-expressing plasmid was included in the transfection mixture.
   
4. Transfection experiments were repeated at least three times.
   
5. Forty-eight hours after transfection, Huh7 cells were incubated at 37 °C in 5% CO₂ incubator for 6 h in 10 ml DMEM containing 10% dialyzed FBS, then labeled at 37 °C in 5% CO₂ for additional 14 h with 250 µCi orthophosphate [³²P_i] per 10⁷ cells, rinsed with ice-cold phosphate-buffered saline, and lysed with 1 ml of lysis buffer. (Isotope was added directly onto the cells containing DMEM-dFBS.)
   
6. Lysates were transferred to 1.5 ml tube, incubated on ice for 10 min for further lysis, spin down at 13,000 rpm at 4 °C for 2 min, and supernatant was transferred to fresh 1.5 ml tube.
   
7. The supernatant lysates were incubated for 2 h at 4 °C with 1 μl of polyclonal rabbit anti-HBc antibody on the rotator.
   
8. Next, 15 μl of protein A/G Plus agarose beads was added, and the mixture was incubated at 4 °C for 1 h and centrifuged at 1,500 rpm for 10 sec. (Protein A/G Plus agarose bead preparation: Take 15 μl of bead/sample to fresh 1.5 ml tube, add 1 ml lysis buffer, vortex briefly, spin down at 1,500 rpm for 10 sec, and discard supernatant. Repeat above washing procedures 2 more times.)
   
9. The pellets in 1.5 ml tube (containing beads bound to immune complexes of radiolabeled WT or mutant C proteins and anti-HBc antibodies) were washed twice with 1 ml lysis buffer, eluted by boiling (100 °C for 5 min) in 15 μl of 2x sample buffer, and separated by SDS-PAGE on 13.5% gels. Running time was 3 h at 80 voltage.
   
10. Separated proteins were transferred to PVDF membranes (250 mA 1 h 30 min) and subjected to autoradiography.
    

Representative data

Compared to AAAAAA mutant of which the phosphoacceptor sites were all abolished to alanine, STSSSS (WT) and other mutants were all ³²Pi-labeled.

Recipes

1. Lysis buffer
   50 mM Tris-HCl (pH 8.5)
   2 mM EDTA
   0.5% Nonidet P-40
   50 mM NaF
   25 mM β-glycerophosphate
   2 mM sodium orthovanadate, and protease inhibitors
   
2. 2x sample buffer
   100 mM Tris-HCl (pH 6.8)
   4% SDS
   200 mM β-mercaptoethanol
   0.2% bromophenol blue
   20% glycerol
   

Acknowledgments

This work was supported by National Research Foundation Grants funded by the Korean Government (NRF-2012-R1A2A2A01015370).

References

1. Jung, J., Kim, H. Y., Kim, T., Shin, B. H., Park, G. S., Park, S., Chwae, Y. J., Shin, H. J. and Kim, K. (2012). C-terminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication. PLoS One 7(7): e41087.
   
2. Jung, J., Hwang, S. G., Chwae, Y. J., Park, S., Shin, H. J. and Kim, K. (2014). Phosphoacceptors threonine 162 and serines 170 and 178 within the carboxyl-terminal RRRS/T motif of the hepatitis B virus core protein make multiple contributions to hepatitis B virus replication. J Virol 88(16): 8754-8767.