1,721,412 research outputs found
Characterization of PF-46396-resistant mutants.
No full text<p>[A] Virus release and CA-SP1 processing in the presence or absence of PF-46396. 293T cells were transfected with WT or mutant pNL4-3 molecular clones, untreated (−) or treated (+) with 5 µM PF-463496 and metabolically labeled with [<sup>35</sup>S]MetCys. Cell- and virus-associated proteins were radioimunoprecipitated. Positions of Pr55<sup>Gag</sup> (PrGag), Pr41<sup>Gag</sup> (p41), CA-SP1 and CA are indicated. [B] Quantification of relative virus release efficiency (VRE) at the indicated concentrations of PF-46396, with release efficiency of WT in the absence of PF-46396 set at 100. Data obtained by phosphorimager analysis of radioimmunoprecipitation data. VRE is calculated as [virus-associated CA+CA-SP1]/[total (cell+virus) Gag]. Error bars indicate SD; N = 3. [C] Quantification of the % CA-SP1 relative to total CA+ CA-SP1 in virion fraction at the indicated concentrations of PF-46396. Error bars indicate SD; N = 3. [D] Quantification of the ratio of cell-associated Pr55<sup>Gag</sup> (p55) to CA+CA-SP1 at the indicated concentrations of PF-46396. Data obtained by phosphorimager analysis of radioimmunoprecipitation data. Error bars denote SD; N = 3.</p
PF-46396 blocks CA-SP1 processing. [A] Chemical structures of BVM and PF-46396.
No full text<p>[B] Radioimmunoprecipitation analysis of virion-associated CA and CA-SP1 in the presence of increasing concentrations of BVM or PF-46396. [C] Quantification of the % CA-SP1 relative to total CA+ CA-SP1 in virion fraction at the indicated concentrations of BVM or PF-46396. Error bars indicate SD; N>3.</p
PF-46396 interferes with BVM activity at low BVM concentration.
No full text<p>293T cells were transfected with WT pNL4-3 and treated with either PF-46396 alone, BVM alone, or PF-46396+BVM at the indicated concentrations. Levels of CA-SP1 in virion fractions were evaluated by radioimmunoprecipitation analysis. Error bars indicate SD; N = 3.</p
Second-site compensatory changes for PF-46396-dependent CA mutants.
No full text<p>Second-site compensatory changes for PF-46396-dependent CA mutants.</p
Structure-activity relationships of the Human Immunodeficiency Virus type 1 maturation inhibitor PF-46396
No full textThis work was funded by the University of St Andrews and Society for Applied Microbiology New Lecturer Research Grant awarded to CSA, Wellcome Trust grant (093228) awarded to TKS.HIV-1 maturation inhibitors are a novel class of antiretroviral compounds, which consist of two structurally distinct chemical classes; betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by a similar mode of action to generate aberrant non-infectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study we utilized a series of novel analogues, with decreasing similarity to PF-46396, to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into WT immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity, but not an absolute requirement for Gag binding, (ii) the trifluromethyl group is optimal but not essential for antiviral activity and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but not essential as its replacement is tolerated.Peer reviewe
Replication kinetics of PF-46396-resistant mutants in the presence or absence of the compound.
No full text<p>[A and B] The Jurkat T-cell line was transfected with WT or mutant pNL4-3 and propagated at the indicated concentration of PF-46396. Virus replication was monitored by RT activity, shown in cpm/µl of culture medium. Note that WT is sensitive to PF-46396; CA-I201V, CA-A1V, and CA-H226Y are resistant; and CA-G156E, CA-P157S, CA-G225D, CA-P160L, SP1-A3V and SP1-A3T are PF-46396-dependent.</p
Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C
No full textAbstractHIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics.</jats:p
PF-46396 resistance mutations map to three regions within the CA-CTD and SP1.
No full text<p>[A] Example of a selection experiment for PF-46396 resistance. The Jurkat T-cell line was transfected with pNL4-3 and propagated in the presence of 0–5 µM PF-46396. Virus replication was monitored by RT activity, shown in counts per minute (cpm) per µl of culture medium. [B] Location of PF-46396 resistance mutations in Gag. A linear diagram of Pr55<sup>Gag</sup> is provided, with CA N-terminal and C-terminal domains (NTD and CTD, respectively) indicated. Small black boxes denote the location in Gag of the resistance mutations, which are shown below using CA and SP1 amino acid numbers. Single-underlined mutations represent those that we previously obtained during BVM selections; highly conserved residues in the MHR are denoted by double underlines.</p
PF-46396 can rescue the assembly/release defect of some but not all MHR mutants.
No full text<p>Top panel: Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence and presence of 5 µM PF-46396. Positions of Pr55<sup>Gag</sup> (PrGag), Pr41<sup>Gag</sup> (p41), CA-SP1, and CA are indicated. Lower panel shows phosphorimager-based quantification of relative virus release efficiency (VRE), calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 3. Note that PF-46396 significantly enhances the release of virus particles for E159Q (and G156E, as shown earlier) but not for Q155N, G156V, or E159D.</p
Second-site compensatory changes correct the replication defects exhibited by PF-46396-dependent CA mutants.
No full text<p>[A] Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence and presence of 5 µM PF-46396. Analysis performed with WT, CA-G156E, CA-N193H, and CA-G156E/N193H. Positions of Pr55<sup>Gag</sup> (PrGag), Pr41<sup>Gag</sup> (p41), CA-SP1, and CA are indicated. Graph on the right shows phosphorimager-based quantification of the % CA-SP1 relative to total CA+ CA-SP1 in virion fraction at the indicated concentration of PF-46396. Error bars denote SD; N = 5. [B] Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence and presence of 5 µM PF-46396. Analysis performed with WT, CA-G157S, CA-P157S/SP1-T8I, CA-G225D, CA-G225D/SP1-T8I, and SP1-T8I. Positions of Pr55<sup>Gag</sup> (PrGag), Pr41<sup>Gag</sup> (p41), CA-SP1, and CA are indicated. Graphs on the right show phosphorimager-based quantification of relative virus release efficiency (VRE) and % virion CA-SP1, calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 3.</p
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