Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells

Materials and Reagents

1. HEK 293T cells
   
2. HeLa cells
   
3. Dulbecco’s Modified Eagle Medium (DMEM) (Mediatech, Cellgro^®, catalog number: 10-013-CV )
   
4. R9-ΔE plasmid ((Zhou and Aiken, 2001), an HIV-1 proviral DNA clone created by introducing a frameshift mutation in envelope of the wild-type infectious R9 clone. Virions produced by this clone are non-infectious but can be made infectious by pseudotyping with envelopes from VSV or other viruses)
   
5. pHCMV-G (VSV-G) plasmid ((Yee et al., 1994), a retrovirus-derived plasmid in which the retroviral envelope glycoprotein is replaced with glycoprotein from vesicular stomatitis virus [VSV]).
   
6. p24 ELISA kit (in-house)
   
7. Phosphate-buffered saline (PBS) (Mediatech, Cellgro^®, catalog number: 21-0310-CV )
   
8. VSV-G-pseudotyped envelope-defective HIV-1 (R9-ΔE) virus particles
   
9. Efavirenz (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
   NIH, catalog number: 11680 )
   
10. DNase I (Roche, catalog number: 10104159001 )
    
11. 0.25% Trypsin/2.21 mM EDTA (Mediatech, Cellgro^®, catalog number: 25-053-CI )
    
12. Triton X-100 (Mallinckrodt, catalog number: 9002-93-1 )
    
13. DNeasy blood and tissue kit (QIAGEN, catalog number: 69506 )
    
14. cOmplete, Mini, EDTA-free protease-inhibitor cocktail tablet (Roche, catalog number: 11836170001 )
    
15. 4 to 20% Polyacrylamide gradient Tris-glycine gels (Bio-Rad)
    
16. Nitrocellulose membrane (General Electric Company)
    
17. Mouse monoclonal anti-GAPDH antibody (Santa Cruz, catalog number: sc-47724 )
    
18. Mouse monoclonal anti-LaminB1 antibody (Life Technologies, catalog number: 33-2000 )
    
19. SYBR green (ABI, catalog number : 4309155 )
    
20. DpnI (New England Biolabs, catalog number: R0176L )
    
21. DTT
    
22. Yeast tRNA (Roche, catalog number : 10109541001 )
    
23. Forward primer MH531 (5’-TGTGTGCCCGTCTGTTGTGT-3’)
    
24. Reverse primer MH532 (5’-GAGTCCTGCGTCGAGAGAGC-3’)
    
25. DNase/RNase-free water
    
26. SDS-PAGE sample buffer
    
27. Sodium deoxycholate (Sigma-Aldrich, catalog number: 30970 )
    
28. N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES) (Sigma-Aldrich, catalog number: B4554 )
    
29. Hypotonic buffer (see Recipes)
    
30. Radioimmunoprecipitation buffer (see Recipes)
    
31. 2x BES-buffered saline (BBS) (see Recipes)
    

Equipment

1. 10 cm cell culture dish
   
2. 0.45-μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2545 )
   
3. 0.20- μm-pore-size syringe filters (Thermo Fisher Scientific, catalog number: 190-2520 )
   
4. 1.5 ml screw-cap tube
   
5. Tabletop centrifuge (Thermo Fisher Scientific, Sorvall^®)
   
6. Tabletop refrigerated centrifuge (Thermo Fisher Scientific)
   
7. Mx-3000p thermocycler (Stratagene)
   
8. CO₂ incubator
   

Procedure