p66 Trp24 and Phe61 Are Essential for Accurate Association of HIV-1 Reverse Transcriptase with Primer/Template

Agopian, Audrey; Depollier, Julien; Lionne, Corinne; Divita, Gilles

doi:10.1016/j.jmb.2007.07.044

Cited by 21 publications

(27 citation statements)

References 44 publications

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“…RT⅐p/t pre-steady-state binding kinetics follow a three-step mechanism in the presence or in the absence of P AW , including a rapid diffusion controlled second order step leading to the formation of the RT⅐p/t collision complex, followed by two slow, concentration-independent, conformational changes (34). The plot of the pseudo-first order rate constant for the initial association of the p/t with RT against RT concentration is linear.…”

Section: P Aw Peptide Interacts With Hiv-1 Rt In a Cellularmentioning

confidence: 99%

“…Expression and Purification of HIV-1 RT Proteins-Histagged RTs were expressed and purified as previously described (23,34). Briefly, M15 bacteria (Qiagen) were separately transformed with all the constructs of p51 and p66 subunits.…”

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

“…Excitation was performed at 492 nm, and emission spectra were recorded from 500 to 600 nm. According to fluorescence experiments, a fixed concentration of FAM-labeled (19/36) p/t (50 nM) or of FITC-P AW (200 nM) was titrated with increasing protein concentrations from 5 nM to 1 M. Data were fitted as previously described (34,37), using a quadratic equation (GraFit, Erithacus Software).…”

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

“…6-Carboxyfluorescein fluorescence was excited at 492 nm, and emission was detected through a filter with a cutoff at 530 nm. Data acquisition and analysis were performed using KinetAsyst 3 software (Hi-Tech Scientific), and traces were fitted according to a three exponential equation, as previously described (34). The rate constant for the first phase (k ϩ1 and k Ϫ1 ), corresponding to the formation of a RT⅐P AW ⅐p/t collision complex, was extrapolated from the slope and the intercept with the y axis of the plot of k obs1 versus RT concentrations.…”

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

“…3A). Effect of P AW Peptide on Primer/Template Binding to HIV-1 RT-The impact of P AW peptide on the ability of HIV-1 RT to bind p/t was then investigated at both steady-state and presteady-state levels using a 19/36-mer p/t labeled at the 3Ј-end of the primer with FAM-derivative as previously described (34,37). As reported in Fig.…”

Section: P Aw Peptide Interacts With Hiv-1 Rt In a Cellularmentioning

confidence: 99%

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A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

Agopian

Gros

Aldrian

et al. 2009

Journal of Biological Chemistry

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The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P AW that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P AW only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P AW prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics. Human immunodeficiency virus type I (HIV-1)4 is the primary cause of AIDS, a slow progressive and degenerative disease of the human immune system. Despite recent therapeutic developments and the introduction of highly active antiretroviral therapy, the rapid emergence of drug-resistant viruses against all approved drugs together with inaccessible latent virus reservoirs and side effects of currently used compounds have limited the efficacy of existing anti-HIV-1 therapeutics (1).Therefore, there is still an urgent need for new and safer drugs, active against resistant viral strains or directed toward novel targets in the replicative cycle, which will be useful for multiple drug combination.HIV-1 reverse transcriptase (RT) plays an essential multifunctional role in the replication of the virus, by catalyzing the synthesis of double-stranded DNA from the single strand retroviral RNA genome (2, 3). The majority of the chemotherapeutic agents used in AIDS treatments target the polymerase activity of HIV-1 RT, such as nucleoside reverse transcriptase inhibitors (NRTI) or non-nucleoside inhibitors (NNRTI) (4). The biologically active form of RT is an asymmetric heterodimer that consists of two subunits, p66 and p51, derived from p66 by proteolytic cleavage of the C-terminal RNase H domain (2, 3, 5).The polymerase domain of both p66 and p51 subunit can be subdivided into four common subdomains: fingers, palm, thumb, and connection (6 -10). Determination of the threedimensional structures of RTs has revealed that, although the folding of individual subdomains is similar in p66 and p51, their spatial arrangement differs markedly (11). The p66 subunit contains both polymerase and RNase H active sites. The p66-polymerase domain folds into an "open," extended structure, forming a large active site ...

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Section: P Aw Peptide Interacts With Hiv-1 Rt In a Cellularmentioning

confidence: 99%

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

Section: Materials-poly(ra)-oligo(dt) and [mentioning

confidence: 99%

Section: P Aw Peptide Interacts With Hiv-1 Rt In a Cellularmentioning

confidence: 99%

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