HIV-1 Single Cycle Infection   

Materials and Reagents

1. HeLa CD4⁺ CCR5⁺ LTR lacZ (also known as P4C5) cells (Verrier et al., 1997)
   
2. Dulbecco Eagle’s Modified Medium (DMEM) (Life Technologies, Gibco^®)
   
3. HIV-1 Δenv viral particles, produced by transfection of HEK-293T cells
   
4. Nonidet P40 (NP40) (Sigma-Aldrich)
   No longer commercially available, but replaced by Igepal CA-630 (Sigma-Aldrich, catalog number: I8896 )
   
5. Phosphate buffer saline (PBS) (Life Technologies, Gibco^®)
   
6. Chlorophenol Red-β-D-Galactopyranoside (CPRG) (F. Hoffmann-La Roche, catalog number: 10884308001 )
   
7. Fetal bovine serum (FBS) (Thermo Fisher Scientific)
   
8. Penicillin/Streptomycin (PS) (Life Technologies, Gibco^®)

Equipment

1. 96-well spectrophotometer
2. Flat-bottom 96-well plates (Corning, Costar^®)
   

Procedure

1. P4C5 cells are HeLa cells, stably expressing CD4 and CCR5. Additionally, they express the lacZ gene under the control of HIV-1 Long Terminal Repeat (LTR) promoter. They are grown like normal HeLa cells, in DMEM, 10% heat-inactivated FBS, 10% PS, and kept at 5% CO₂ and at 37 °C.
   
2. 8 x 10⁴ P4C5 cells were plated in flat-bottom 96-well plates in a final volume of 100 μl of complete medium for 24 h.
   
3. Cells were infected in triplicate with HIV-1 Δenv, using 1 or 5 ng of Gag p24 per well. Specifically, 100 μl of virus-containing medium were added to the 100 μl of complete medium already in the well. Viral titers have to be previously determined using a Gag p24 ELISA assay (either home-made or commercial). The use of two different viral inputs is required to make sure that the saturation levels are not reached.
   
4. At 36 h post-infection, cells were lysed in 80 μl of lysis buffer, then incubated for 1 min at room temperature with 80 μl of lysis buffer, containing CPRG at a final concentration of 3.65 mg/ml. CPRG, the substrate of the β-galactosidase enzyme, allows to monitor the levels of infection. OD (570 nm) was measured every 15 min and normalized against background levels of OD (690 nm).
   
5. Infection levels - as monitored by percentage of Gag⁺ cells by flow cytometry, or quantification of Gag p24 in the culture supernatants by ELISA - are proportionnal to the normalized OD (570 nm). The assay is linear from 0.1 to 3 units of OD (570 nm). Representative results are shown in Figure 3B of Roesch et al. 2012.
   

Recipes

1. Complete medium
   DMEM
   10% heat-inactivated FBS
   10% PS
   
2. Lysis buffer
   PBS
   0.1% NP40
   5 mM MgCl₂
   

Acknowledgments

This protocol is adapted from Roesch et al. (2012).

References

1. Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.
   
2. Verrier, F. C., Charneau, P., Altmeyer, R., Laurent, S., Borman, A. M. and Girard, M. (1997). Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate. Proc Natl Acad Sci U S A 94(17): 9326-9331.