Figure 2.

Experimental strategy to quantify in vitro HIV-1 replication in PBMC. (A) Simplified schema of the replication-competent R5-tropic HIV-1 reporter virus vNL-sNLuc.6ATRi-B-Bal.ecto. The vector was based on the NL-Bal.Env.ecto-derived molecular clone of HIV-1 expressing Renilla luciferase previously generated by Ochsenbauer and Astronomo et al. [50,51]. The reporter gene (indicated as the black area) is a secreted form of nanoluciferase (encoded by the reporter gene sNLuc); nef expression is regulated by an internal ribosome entry site (IRES). The sNLuc.IRES cassette was inserted between the NL.Bal.ecto env and nef genes; 6ATRi is a truncation (“TR”) fragment derived from the encephalomyocarditis virus (EMCV) IRES and contains the “wild type” (A)₆ (i.e., “6A”) bifurcation loop. The nucleotide sequence shows the junction between the stop codon of env (taa) and the start codon (atg) of sNLuc, and contains a Nhe I restriction site (gct agc) and the translation initiation Kozak sequence (ccacc). The construct contains a 26 nt “IRES spacer” between the sNLuc gene and the IRES element, as previously described [51]. (B) Example of an in vitro HIV-1 infection experiment. PBMC were infected at MOI 0.2 in the presence (empty circles) or absence (full circles) of 1 µM raltegravir and 1 µM efavirenz. Aliquots from supernatants were taken at the indicated time points (x-axis) and nanoluciferase activity was determined using the Promega Nano-Glo© kit (y-axis, RLU: relative light units).