Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA

Materials and Reagents

1. Huh-7.5 cells (obtained from Apath, LLC) expressing TLR3-Flag (or other cells stably/transiently expressing Flag/Myc-tagged protein)
   
2. DMEM (Life Technologies, catalog number: 11995065 )
   
3. 10% heat-inactivated FBS (Life Technologies, catalog number: 26140079 )
   
4. Penicillin-streptomycin (Life Technologies, catalog number: 15140-148 )
   
5. L-Glutamine (Life Technologies, catalog number: 25030-081 )
   
6. Non-essential amino acids (Life Technologies, catalog number: 11140050 )
   
7. HCV strain HJ3-5 stock prepared in cell culture medium (see Reference 2)
   
8. DPBS (Life Technologies, catalog number: 14190-144 )
   
9. RNase inhibitor (RNaseOUT) (Life Technologies, catalog number: 10777-019 )
   
10. Protease inhibitor (Complete Protease Inhibitor Cocktail Tablets) (Roche Diagnostics, catalog number: 1697498 )
    
11. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
    
12. Anti-Flag (M2) and anti-Myc (9E10) monoclonal antibodies (Sigma-Aldrich, catalog numbers: F1804 and M4439 ) and mouse IgG control (Life Technologies, catalog number: 02-6502 )
    
13. Protein G sepharose (General Electric Company , catalog number: 17-0618-01 )
    
14. TRIzol (Life Technologies, catalog number: 15596-026 )
    
15. Molecular biology grade water (Corning, catalog number: 46-000-CM )
    
16. Agarose (GenePure LE Agarose) (BioExpress, GeneMate, catalog number: E-3120-500 )
    
17. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
    
18. Platinum^® Taq DNA Polymerase (Life Technologies)
19. Culture medium (see Recipes)
    
20. Lysis buffer (see Recipes)
    

Equipment

1. 100 mm plates (Falcon^TM, catalog number: 353003 )
   
2. Cell scraper (Falcon^TM, catalog number: 353085 )
   
3. Electrophoresis Gel Box
   
4. Tube Rotator (Fisher Scientific, catalog number: 05-450-200 )
   
5. Centrifuge (Eppendorf, catalog number: 5415R )
   
6. Superscript III One-step RT-PCR system (Life Technologies, catalog number: 12574-018 )
   

Procedure

1. Preparation of HCV infected cells
   1. Seed the Huh-7.5 cells (1.5 x 10⁶ per dish) stably expressing TLR3-Flag onto a 10 cm dish and incubated overnight.
      
   2. Inoculate HCV (strain HJ3-5) at an MOI of 1 (5 ml of HJ3-5 stock at 3 x 10⁵ FFU/ml) for 6 h, remove the inoculum, and then replace with 10 ml fresh culture medium.
      
   3. Incubate cells at 37 °C in 5% CO₂ for 72 h.
      
      
2. Preparation of the lysates
   1. Aspirate culture medium, wash cells twice with DPBS, and scrape the cells into 50 ml tube.
      
   2. Centrifuge at 800 x g for 3 min at 4 °C, and remove the supernatant.
      
   3. Resuspend the cell pellet in 1 ml lysis buffer, and rotate the lysate for 5-6 h at 4 °C.
      
   4. Centrifuge the lysate at 15,700 x g for 20 min at 4 °C.
      
   5. Transfer the supernatant to 1.5 ml tube and place on ice, and determine the protein content using Protein Assay Kit.
      
      
3. Immunoprecipitation
   1. Transfer the cell lysate (20 μg of total protein) to a new 1.5 ml tube.
      
   2. Add 1 μg of anti-Flag antibody or mouse IgG control to the lysate and rotate overnight at 4 °C.
      
   3. Add 20 μl Protein G sepharose to the lysate and rotate for 2-3 h at 4 °C.
      
   4. Centrifuge at 800 x g for 3 min at 4 °C.
      
   5. Aspirate the supernatant, wash beads with 1 ml lysis buffer and rotate for 10 min at 4 °C.
      
   6. Centrifuge at 800 x g for 3 min at 4 °C.
      
   7. Repeat steps C4-5 twice.
      
   8. Suspend the sepharose in 100 μl DPBS and use half for RNA extraction and the remainder for Western blotting to detect the immunoprecipitated protein.
      
      
4. RNA extraction and RT-PCR
   1. Extract RNA from the sepharose beads by vortexing 15 sec in TRIzol reagent, followed by the standard protocol as indicated in the manufacturer’s instruction, and suspend the RNA pellet in 50 μl of nuclease-free water (optional: Add 1 μl of Glycogen before the precipitation of RNA with Isopropanol).
      
   2. Detect HCV RNA bound to TLR3-Flag with SuperScriptIII One-Step RT-PCR System with Platinum^® Taq DNA Polymerase using an HCV-specific primer pair HCV84FP, 5’-GCCATGGCGTTAGTATGAGTGT-3’; HCV 303RP, 5’-CACCCTATCAGGCAGTACCACAA-3’, at an annealing temperature of 55 °C, followed by gel electrophoresis in 1.5% agarose gel. Specific bands (220 bp) can be detected typically with 35-40 PCR cycles.
      

Recipes

1. Culture medium
   DMEM
   10% heat-inactivated FBS
   Penicillin-streptomycin
   L-Glutamine
   Non-essential amino acids
   
2. Lysis buffer
   1x DPBS
   0.1% Triton X-100
   1x Protease inhibitor cocktail
   100 U/ml RNaseOUT
   

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (RO1-AI095690) and the University Cancer Research Fund. This protocol is adapted from previous work by Dansako et al. (2013).

References

1. Dansako, H., Yamane, D., Welsch, C., McGivern, D. R., Hu, F., Kato, N. and Lemon, S. M. (2013). Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9(5): e1003345.
   
2. Yi, M., Ma, Y., Yates, J. and Lemon, S. M. (2007). Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81(2): 629-638.