Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol

Abstract

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation. HIV-1 encodes reverse transcriptase (RT), an enzyme that is essential for virus replication. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 RT. In HIV-1-infected cells NNRTIs block the RT-catalyzed synthesis of a double-stranded DNA copy of the viral genomic RNA, which is an early step in the virus life cycle. Potent NNRTIs have the novel feature of promoting the interaction between the two RT subunits. However, the importance of this effect on the inhibition of HIV-1 replication has not been defined. In this study, the authors show that potent NNRTIs block an additional step in the virus life cycle. NNRTIs increase the intracellular processing of viral polyproteins called Gag and Gag-Pol that express the HIV-1 structural proteins and viral enzymes. Enhanced polyprotein processing is associated with a decrease in viral particles released from NNRTI-treated cells. NNRTI enhanced polyprotein processing is likely due to the drug binding to RT, expressed as part of the Gag-Pol polyprotein and promoting the interaction between separate Gag-Pol polyproteins. This leads to premature activation of the Gag-Pol embedded HIV-1 protease, resulting in a decrease in full-length viral polyproteins available for assembly and budding from the host cell membrane. This study provides proof-of-concept that small molecules can modulate the interactions between Gag-Pol polyproteins and suggests a new target for the development of HIV-1 antiviral drugs.