Dead-end complexes contribute to the synergistic inhibition of HIV-1 RT by the combination of rilpivirine, emtricitabine, and tenofovir

•Combinations of rilpivirine, emtricitabine, and tenofovir display anti-HIV-1 synergy.•Gel shift assays show dead-end complex (DEC) formation with these drugs.•Mechanisms of synergy map to DEC-like complexes.

The single tablet regimen of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and the non-nucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) is approved for the treatment of HIV-1 infection in treatment-naïve adults. Previous studies have shown that two-drug combinations of these drugs show additive to synergistic HIV-1 antiviral activity in cell culture. In this study, two-drug combinations of tenofovir (TFV) + FTC, RPV + TFV, and RPV + FTC inhibited HIV-1 replication in cell culture with strong synergy and no evidence of antagonism. The triple drug combination of RPV + FTC + TFV displayed moderate synergy comparable to efavirenz (EFV) + FTC + TFV. The formation of dead-end complexes (DEC) of HIV-1 reverse transcriptase (RT), NRTI chain-terminated primer/template, and the next complementary nucleotide or NNRTIs was studied using gel mobility shift assays. DEC formation was seen with TFV-terminated DNA primer/template, HIV-1 RT, and FTC-triphosphate (TP) in addition to the natural nucleotide dCTP, thus stabilizing chain-termination. The NNRTI RPV also formed DEC-like complexes with TFV- and FTC-monophosphate (MP)-terminated DNA primer/templates and HIV-1 RT, and stabilized chain-termination by both NRTIs. Overall, the combinations of RPV, FTC, and TFV inhibit HIV-1 replication with moderate to strong synergy. This may be partially explained by enhanced DEC formation of NRTI chain-terminated DNA primer/template and HIV-1 RT in the presence of the other drugs in the combination, leading to more stable chain-termination and replication inhibition by NRTIs.