Second-generation Maturation inhibitors (BVM analogs) were synthesized by chemical modifications at C-28 positions of Bevirimat (BVM) by DFH Pharma, USA. All the compounds were clean and consistent and showed 99% purity by LC/MS. Compounds were dissolved in dimethyl sulfoxide (DMSO) and stored in the dark in − 80 °C freezer.

HEK-293 T and HUT-R5 T-cells were kind gifts from Dr. Eric O. Freed, National Cancer Institute, NIH, USA. TZM-bl cells (catalog number: 8129) were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. HEK-293 T and TZM-bl cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM). HUT-R5 T-cells were maintained in (RPMI)-1640 (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

HIV-1 subtype B clone NL4-3 (a kind gift from Dr Eric O. Freed, National Cancer Institute, NIH, USA; GenBank accession: AF324493.2), HIV-1 subtype C clone; K3016 (South African origin, a kind gift from Dr. Christina Ochsenbauer, University of Alabama, USA; GeneBank accession: KC156129), Protease inhibitor-resistant and Integrase inhibitor-resistant clones from the National Institutes of Health AIDS Reagent Program(NIH ARP 11800 and NIH ARP 11847 respectively) [13] were used in this study.

HEK-293 T cells were transfected with 3 μg HIV-1 DNAs (HIV-1 subtype B clone NL4-3, HIV-1 subtype C clone K3016 WT and protease-resistant and integrase resistant mutant clone, NL4-3 11,800 and NL4-3 11,847 respectively) using Lipofectamine 2000 (Invitrogen catalog number: 11668-019).

HEK-293 T cells grown in six-well plates to about 80% confluency were transfected with HIV-1 DNAs (3 μg). The culture medium was replaced with fresh DMEM after 24 h post-transfection and incubated for another 2 h. MIs were maintained in the culture throughout transfection. The viral supernatants were filtered using a 0.45 μm pore size filter disc to remove residual cellular contaminants. The virus was pelleted by ultracentrifugation at 210,100 × g for 1 h at 4 °C using a SW41Ti rotor (Beckman Coulter, USA). To measure the accumulation of CA-SP1, viral lysates were subjected to SDS–polyacrylamide gel electrophoresis (15% gel); proteins were transferred to polyvinylidene difluoride membranes and reacted with HIV-IgG (NIH AIDS Reagent Program; catalog no. 3957) followed by incubation with HRP-conjugated anti-human secondary antibodies (GE Healthcare, UK). The proteins were visualized by enhanced chemiluminescence (BioRad, USA), and the bands were quantified using ImageJ software (http://imagej.nih.gov/ij/).

Cytotoxicity assays were performed using the CellTitre-Blue Cell Viability Assay kit (Promega, USA) as per the manufacturer’s recommendations. HEK-293 T and HUT-R5 T-cells were maintained in the presence of serial dilutions of MIs for 48 h and treated with CellTitre-Blue reagent for 4 h at 37 °C. The fluorescent signals were recorded at 530/25 excitation and 590/35 emission using a BioTek microplate reader. The 50% cytotoxicity concentrations (CC50s) were determined as the concentrations of MI that reduced the fluorescent signals to 50% relative to DMSO-only controls.

1.6 × 106 HUT-R5 T-cells were infected with p24-normalized HIV-1 subtype C K3016 virus (50 ng p24 equivalent) stocks at 37 °C for 2 h. 2 × 104 virus infected cells were then cultured in 96 well plate in the presence of serial dilutions (500 nM,100 nM,20 nM,4 nm and 0.8 nM)of each compound. The virus supernatants were collected after 8 days, and the concentration of HIV-1 p24 was measured in the virus supernatants. Compared to DMSO-only controls, the doses of MI that decreased HIV-1 p24 concentrations to 50% were determined to be the 50% inhibitory concentrations (IC50).

IC50 analyses using single-cycle infectivity assays were also performed. HEK-293 T cells were transfected with K3016 and cultured in the presence of serial dilution of MIs. Virus-containing supernatants were harvested, normalized for HIV-1p24 concentration, and used to infect TZM-bl cells. Luciferase activity was measured two days post-infection, and infectivity was calculated. The 50% inhibitory concentrations (IC50) were calculated as the concentrations of MI that reduced infectivity to 50% relative to DMSO-only control using the GraphPad Prism V5.0.

HEK-293 T cells were transfected with HIV-1 DNAs (HIV-1 subtype B clone NL4-3, HIV-1 subtype C clone K3016 WT, and mutants NL4-3 11,800 and NL4-3 11,847. DMSO or maturation inhibitors (MIs) were maintained in the culture throughout transfection. 24 h post-transfection, culture supernatant was collected and filtered using a 0.45 μm pore size filter disc to remove residual cellular contaminants. The virus stocks were normalized for p24 antigen using an XpressBio ELISA kit (Catalog number: XB-1000). 5 ng HIV-1 p24 equivalent virus was used to infect TZM-bl cells (5 × 104/well) in the presence of 20 μg DEAE-dextran per ml in 24 wells plate. The luciferase activity in the cell lysates was measured 48 h post-infection using the Steady-Glo luciferase assay kit (Promega catalog number: E2510).

All statistical analysis was performed using GraphPad Prism V5.0. One-way ANOVA was done by implying Tukey’s test. p values below 0.05, 0.01,0.001 and 0.0001 were denoted as *, **, *** and ****, respectively. Non-significant results were denoted as ns.