Olution and was added to the culture medium at a final
Olution and was added to the culture medium at a final concentration as indicated. The equivalent volume of DMSO was added to control cultures.HTRF ased INLEDGF interaction assaywas determined, after 5 or 7 days for HIV-1 and SIV respectively, using the CellTiter-Glo?luminescent reagent (Promega) to quantify cell viability.Viral resistance selectionThe IN-LEDGF HTRF?assay was performed using Flag-tagged IN NL4-3 and His-tagged LEDGF/p75 as described in [12], and detailed in Additional file 1.HTRF ased IN multimerization assayThe IN N HTRF?assay was performed using Flagtagged and His-tagged IN from NL4-3, as described in [12] and detailed in Additional file 1.Virus production and replicationThe experiments with HIV-1 isolates LAI or NL4-3 were performed as described in [23] and the data presented here were collected simultaneously with those reported previously in [23] using the INLAI BI-D together with the same untreated controls. 293T cells were seeded in T75 culture flasks, cultured to 50?0 confluency and transfected with 20 g pLAI or pNL4-3 DNA plasmid that encodes the wt HIV-1 LAI or HIV-1 NL4-3 isolates respectively using Lipofectamine2000 (Invitrogen). MUT-A was added 6 h after transfection. The culture supernatant was harvested 48 h after transfection and used as virus stock or for viral RNA isolation. The CA-p24 level was measured by enzyme-linked immunosorbent assay (ELISA) as described previously [23]. SupT1 T cells (5 ?106 cells in 5 mL) were infected with the HIV-1 LAI virus stocks (equivalent of 1 ng CA-p24). Similarly, MT4 cells (5 ?106 cells in 5 mL) were infected with the HIV-1 NL4-3 virus stocks (equivalent of 1 ng CA-p24). When indicated, the culture was split and MUT-A or DMSO was added. Viral spread was monitored by measuring the CA-p24 level in the virus culture medium every 2 days. HIV-1 NL4-3 virus produced and inactivated in the presence of AT-2 was prepared as described in [24].HIV1 and SIV antiviral assays in MT4 cellsResistance selection was carried out by serial passages of infection at suboptimal compound concentrations. MT4 cells were seeded in 96-well culture plates at a density of 1 ? 105 cells/well in 200 of culture medium. HIV-1 NL4-3 was used at 0.005 MOI. Compounds were added at final concentrations corresponding to ?EC50, EC50 and 2? EC50 and the plates were done in triplicate. Every 3? days, measurement of the antiviral A-836339 site activity allowed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 selecting between the three successive concentrations which one to choose to go on with the next passage. The cytopathic effect induced by HIV-1 was used to follow the progression of the infection. Briefly, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26437915 50 of the cell culture from the passage plate were mixed with 50 CellTiterGlo?reagent to quantify cell viability. The concentration corresponding to the lower protection (0?5 ) at the highest concentration was chosen to proceed. All the content from the chosen well was transferred to a 1.5 mL microtube and centrifuged for 3 min at 3000 rpm at room temperature. 30 of the supernatant were then used to infect three wells seeded with fresh cells and twofold serial dilutions of the compound. Successive viral passages were obtained by repeating this procedure.Clonal sequencing of viral DNAMT4 cells growing exponentially at the density of 106/mL were infected for 2 h with HIV-1 strain NL4-3, HxB2 or SIVmac239 (viral stock produced according to [25]) at a multiplicity of infection (MOI) of 0.006 and 0.01, respectively. The cells were washed with PBS and.