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

Citation: Figueiredo A, Moore KL, Mak J, Sluis-Cremer N, de Bethune MP, et al. ( Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol Anna Figueiredo 0 Katie L. Moore 0 Johnson Mak 0 Nicolas Sluis-Cremer 0 Marie-Pierre de Bethune 0 Gilda Tachedjian 0 0 1 Molecular Interactions Group, Macfarlane Burnet Institute for Medical Research and Public Health , Melbourne , Australia , 2 Department of Microbiology, Monash University , Clayton , Australia , 3 HIV Assembly Group, Macfarlane Burnet Institute for Medical Research and Public Health , Melbourne , Australia , 4 Department of Biochemistry and Molecular Biology, Monash University , Clayton , Australia , 5 Department of Medicine, University of Pittsburgh School of Medicine , Pittsburgh , Pennsylvania, United States of America , 6 Tibotec Pharmaceuticals, Mechelen , Belgium , 7 Department of Medicine, Monash University , Prahran , Australia 1 www.plospathogens.org 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. - The HIV-1 reverse transcriptase (RT) is responsible for the conversion of the viral single-stranded genomic RNA into a double-stranded proviral DNA precursor. This process is catalyzed by the RNA- and DNA-dependent polymerase and ribonuclease H activities of the enzyme. HIV-1 RT is an asymmetric dimer that consists of a 66- (p66) and a p66derived 51-kDa (p51) subunit [1]. The RT heterodimer is the biologically active form of the enzyme; monomeric subunits are devoid of polymerase activity [2,3]. The HIV-1 RT is translated as part of a 160-kDa Gag-Pol polyprotein (Pr160gag-pol), which consists of the gag-encoded structural proteins matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and the pol-encoded viral enzymes, the protease (PR, p10), reverse transcriptase (RT, p66/p51), and integrase (IN, p31). A transframe region links the Gag and Pol domains and consists of an N-terminal octapeptide and p6pol [4]. The pol open reading frame partially overlaps with gag and is translated by a ribosomal frameshifting mechanism, which occurs in one out of 20 Gag translation events [5]. This ensures the strict maintenance of a 20:1 ratio of Gag to Gag-Pol that is important for viral assembly, replication, and the production of infectious virions [6]. During or subsequent to virus budding, the viral PR autoactivates and cleaves Gag and Gag-Pol into the structural and viral proteins, which results in the maturation of immature particles to form infectious virions [7]. While HIV-1 PR activation is a critical step in the viral life cycle, the processes required for PR activation in HIV-1infected cells is not well defined [7,8]. It is thought that GagPol multimerization during viral assembly leads to activation of the HIV-1 PR by dimerization of PR regions on separate Gag-Pol polyproteins, followed by the autocatalytic cleavage and release of a functionally active PR homodimer [7]. Although direct multimerization of Gag-Pol has not been demonstrated biochemically, several domains within Gag-Pol have been shown to influence PR activation including regions that are proximal to the C- and N-termini of PR [913]. If Gag-Pol dimerizes, as predicted, then HIV-1 RT, due to its size and propensity to dimerize, is likely to contribute to GagPol dimerization and promote PR activation. In support of this notion, deletions or C-terminal truncations of the RT in the context of Gag-Pol leads to decreased processing of Gag and Gag-Pol and impaired virus maturation [9,11,14]. Therefore, the proper regulation of Gag and Gag-Pol processing is an essential step in the production of mature viral particles. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically diverse group of lipophilic compounds that comprise over 30 different classes and specifically inhibit HIV-1, but not HIV-2 RT [15]. NNRTIs bind to an allosteric 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. pocket in the p66 subunit of the RT and inhibit DNA synthesis reactions by a non-competitive mechanism of action [16,17]. Currently, three NNRTIs, namely nevirapine (NVP) [18], delavirdine (DLV) [19], and efavirenz (EFV) [20] have been approved for the treatment of HIV-1. However, the genetic threshold (...truncated)