Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

Abstract

d Efavirenz (EFV), a nonnucleoside reverse transcriptase (RT) inhibitor, also inhibits HIV-1 particle release through enhanced Gag/Gag-Pol processing by protease (PR). To better understand the mechanisms of the EFV-mediated enhancement of Gag processing, we examined the intracellular localization of Gag/Gag-Pol processing products and their precursors. Confocal microscopy revealed that in the presence of EFV, the N-terminal p17 matrix (p17MA) fragment was uniformly distributed at the plasma membrane (PM) but the central p24 capsid (p24CA) and the Pol-encoded RT antigens were diffusely distributed in the cytoplasm, and all of the above were observed in puncta at the PM in the absence of EFV. EFV did not impair PM targeting of Gag/ Gag-Pol precursors. Membrane flotation analysis confirmed these findings. Such uniform distribution of p17MA at the PM was not seen by overexpression of Gag-Pol and was suppressed when EFV-resistant HIV-1 was used. Forster's fluorescence resonance energy transfer assay revealed that Gag-Pol precursor dimerization occurred mainly at the PM and that EFV induced a significant increase of the Gag-Pol dimerization at the PM. Gag-Pol dimerization was not enhanced when HIV-1 contained the EFV resistance mutation in RT. Bacterial two-hybrid assay showed that EFV enhanced the dimerization of PR-RT fragments and restored the dimerization impaired by the dimerization-defective mutation in the RT tryptophan repeat motif but not that impaired by the mutation at the PR dimer interface. Collectively, our data indicate that EFV enhances Gag-Pol precursor dimerization, likely after PM targeting but before complete particle assembly, resulting in uniform distribution of p17MA to and dissociation of p24CA and RT from the PM.

Nonnucleoside Reverse Transcriptase Inhibitors Reduce HIV-1 Production from Latently Infected Resting CD4 + T Cells following Latency Reversal

“…Collectively, these data reveal that the NNRTIs EFV and RPV significantly attenuate the kick of latent HIV-1 from resting CD4 ϩ T cells by inhibiting the release of HIV-1 virus particles. This finding is consistent with our prior work, which demonstrated that potent NNRTIs impact the late stages of HIV-1 replication (13), leading to a decrease in virus production from HIV-1-transfected 293T or HeLa cells (14,15). Specifically, NNRTIs enhance Gag-Pol polyprotein precursor dimerization, likely after plasma membrane targeting but before complete particle assembly, resulting in uniform distribution of p17 matrix to and dissociation of p24 capsid and reverse transcriptase from the plasma membrane (13)(14)(15).…”

“…This finding is consistent with our prior work, which demonstrated that potent NNRTIs impact the late stages of HIV-1 replication (13), leading to a decrease in virus production from HIV-1-transfected 293T or HeLa cells (14,15). Specifically, NNRTIs enhance Gag-Pol polyprotein precursor dimerization, likely after plasma membrane targeting but before complete particle assembly, resulting in uniform distribution of p17 matrix to and dissociation of p24 capsid and reverse transcriptase from the plasma membrane (13)(14)(15). Interestingly, in the HeLa and 293T cells, micromolar concentrations of EFV were required to see a significant reduction in virus production (14,15).…”

“…Specifically, NNRTIs enhance Gag-Pol polyprotein precursor dimerization, likely after plasma membrane targeting but before complete particle assembly, resulting in uniform distribution of p17 matrix to and dissociation of p24 capsid and reverse transcriptase from the plasma membrane (13)(14)(15). Interestingly, in the HeLa and 293T cells, micromolar concentrations of EFV were required to see a significant reduction in virus production (14,15). In contrast, the concentration of either EFV or RPV required to decrease virus production from resting CD4 ϩ T cells was in the nanomolar range ( Fig.…”

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

“…These results are consistent with the studies summarized in this review, indicating that processing of the p51-RH bond is dependent on RH unfolding, which in turn depends on dimer formation. Experimental studies supporting the cooperativity of dimer formation have been reported by Figueiredo et al [87] and Sudo et al [117], who showed that the addition of EFV or other dimer-inducing NNRTIs to HIV-transfected cells led to premature PR activation, resulting in intracellular processing of the polyprotein and a decrease in viral particle production. In this case, the order of dimer formation differs from that illustrated in Figure 16.…”

“…This type of arrangement also shields potential cleavage sites in the folded domains from HIV protease and form proteases present in the host cell. Dimerization of the polyprotein is facilitated by anchoring of the myristoylated polyprotein in the membrane [116,117], and becomes more probable at the higher concentrations present in the virion—initial concentrations of Gag and Gag-pro-Pol are estimated as 3.6 mM and 0.2 mM, respectively [118], facilitating the formation of the active HIV protease dimer necessary for viral maturation.…”