Presentation of the Entry/Uncoating assay based on core-packaged RNA availability and Translation (EURT).

<p>An entry/uncoating reporter mRNA coding Luciferase (EU-repRNA) was engineered by appending a minimal HIV-1 packaging sequence (Ψ) upstream of the <i>Firefly luciferase (F-Luc</i>, followed by a polyA signal). By virtue of its Ψ sequence, this mRNA behaves as genomic viral RNA and is therefore encapsidated into virion particles and then protected inside viral cores. When HIV-1 virions bearing this reporter are used to challenge target cells, the availability of EU-repRNA to the cytoplasmic translation machinery will be in large measure influenced by the fate and status of the viral core. The cartoon depicts the possible fates of viral cores upon entry, according to data published in the field concerning the early phases of the life cycle of HIV. SL: stem loop; numbers refer to the HIV-1 sequence #K02013. EU-repRNA expression is obtained upon DNA transfection in virus-producing cells and is driven by a CMV promoter (the phase of virion production is not detailed for simplicity).</p

Comparison of the EURT assay with the gold standard in HIV entry, the Blam-Vpr assay.

<p>To compare the EURT assay with the widely used Blam-Vpr assay, virions were produced by transfection of HEK293T cells using either reporters. A) Viruses were produced with an R5- or an X4- tropic Env (JR-FL and NL4-3, respectively), then used to challenge SupT1 and monocyte-derived dendritic cells (MDDCs, derived upon incubation of blood monocytes with GM-CSF and IL4 for 4 days). Envelope-receptor specificity was then measured according to the two viral entry assays in lymphocytes (SupT1) and in myeloid cells (MDDCs) either after cell lysis and luciferase activity measurement, or following the incubation of target cells with the fluorescent dye CCF2 and flow cytometry. B) SupT1 were used as target cells to compare the inhibitory rates that could be retrieved upon usage of Vpr-Blam- or EU-repRNA bearing HIV-1 X4 particles in the presence of the HIV-1 fusion inhibitor T20. The graphs present averages and SEM obtained with 3 independent experiments. * p≤0.05 after a Student t test between WT and the respective indicated conditions.</p

At high concentrations, the HIV-1 core-destabilizing compound PF74 induces an increase in Luciferase activity measured upon EURT assay.

<p>A) Due to its well described destabilizing effect on viral cores, PF74 is expected at doses superior to 10 μM to increase the availability of EU-repRNA to the translation machinery. B) To exclude pleiotropic effects of PF74 on translation, HeLaP4 cells were transfected with an <i>in vitro</i> synthesized, capped mRNA coding F-Luc in the presence or absence of PF74 (at 10 μg/mL, which corresponds to 23 μM). Luciferase accumulation was measured 8 hours afterwards. C and D) HeLa P4 or MDDCs were challenged with HIV-1 virus, bearing either an X4- or an R5-tropic Env and incorporating EU-repRNA (MOI equivalent of 0.5, after exo-RT normalization against standards of known infectious titer) in the presence or absence of PF74 (as above), prior to cell lysis and Luciferase activity measurement. Averages and SEM of 6 independent experiments are presented (with cells obtained from different donors in the case of MDDCs). *statistically significant differences following a Student t test (p≤0.05).</p

Luciferase accumulation following the EURT assay can be modulated by the status of the viral core.

<p>A) Simplified representation of the activity of Owl monkeys Trim-Cyp on HIV-1 capsids after their entry into target cells. B) HIV-1 particles were used to challenge CRFK cells stably expressing or not Trim-Cyp. Given that they lack the appropriate HIV receptors, CRFK cells were priorly transfected with DNAs coding CD4 and CXCR4. C) Scheme of the Gag mutants used here. NL4-3 Env+ and EU-repRNA+ HIV-1 derived viruses were produced by transient transfection of HEK293T cells using the indicated structural proteins. Virions were then purified by ultracentrifugation and normalized by protein content. D) EU-repRNA incorporation into viral particles was determined by lyzing protein normalized virions, followed by nucleic acid transfer onto a nylon membrane, hybridization with a ³²P-labelled specific probe and phosphor imager quantification. E) The same amount of viral particles used in D was then used to challenge HeLaP4 cells in an EURT assay. The graphs present data obtained with 3 independent experiments. *statistically significant differences following a Student t test (p≤0.05).</p

Contribution of reverse transcription to the susceptibility of viral cores to high doses of PF74.

<p>A) Dual reporter viral particles were produced by co-transfection of HEK293T cells with DNAs coding Gag-Pol, Env along with the two genomes: EU-repRNA and pRRL-GFP that contains all the elements required for its mobilization in target cells. To favor co-packaging of EU-repRNA in reverse transcription competent viral particles, a ratio of 9 to 1 was used between pRRL and EU-repRNA. <i>CMV</i>, cytomegalovirus promoter; <i>LTR</i>, long terminal repeats; <i>Ψ</i>, packaging sequence; <i>PBS</i>, primer binding sequence; <i>RRE</i>, Rev-responsive element; <i>cPPT-CTS</i>, central polypurine tract-central termination sequence; <i>PPT</i>, polypurine tract. B) Dual reporter viral particles containing a wild type or a mutated RT (Q151N) were used to challenge HeLaP4 cells in the presence or absence of Nevirapine (10 μM). PF74 was added at 23 μM at the indicated time post infection and the overall amount of luciferase activity was measured at ten hours post infection. Values are normalized for the ones observed in the absence of PF74. The graphs present AVG and SEM obtained from 4 to 9 independent experiments. *, statistically significant differences following a Student t test (p≤0.05). C) The graph presents the decrease in the amount of PF74-induced luciferase activity in the individual experiments that compose Fig 8B at t3 and t4. D) The graph presents the percentage of PF74-induced luciferase activity lost in reverse transcription competent viruses over reverse transcription incompetent ones (data of Fig 8B, pooling t3 and t4 data points).</p

Primary features of the EURT assay.

<p>A and B) Reporter HIV-1 virion particles were produced by transient transfection of HEK293T cells with DNAs coding the structural proteins Gag-Pro-Pol and Env (NL4-3), along with EU-repRNA. Supernatants were then ultracentrifuged through a 25% (w/v) sucrose cushion and then used to challenge HeLaP4 cells that bear the appropriate HIV-1 receptor and co-receptor (CD4 and CXCR4). Infection was synchronized and F-Luc activity measured 8 hours post challenge, as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005897#ppat.1005897.g001" target="_blank">Fig 1A</a>) As control, transfections in which one viral element was omitted were also similarly prepared and used on target cells (Δ, as indicated). To exclude the presence of protein carry-over contaminating the viral preparations, cells were also challenged with WT virus in the presence of the translation inhibitor Puromycin (10 μg/mL). B) To assess the linearity of this assay, cells were challenged with two-fold dilutions of WT HIV-1 reporter virus (provided at MOI-equivalents comprised between 0.0025 and 1, as determined after exo-RT comparison against standards of known infectious titer). Dotted line and R2 value: linear regression analysis of the results. The small graph on the right expands data obtained with lower viral inputs. All graphs present averages and SEM obtained with 4 to 6 independent experiments. * p≤0.05 after a Student t test between WT and the indicated conditions.</p

Determination of the effects of different concentrations of PF74 on EURT and viral infectivity.

<p>Dual reporter viruses were used to challenge HeLaP4 cells and either left untreated or treated at t0 with the indicated concentration of PF74. The extent of luciferase activity was measured eight hours post infection (A), while the percentage of GFP-positive cells was determined three days later by flow cytometry (B). The graph presents data obtained from 5 independent experiments.</p

Time dependent accumulation of Luciferase over time and influence of Nevirapine and different concentrations of PF74.

<p>A) To determine the extent to which reverse transcription could expose the viral genome and therefore increase the accumulation of Luciferase activity, dual reporter and reverse transcription competent viral particles were used to challenge HeLaP4 cells according to the scheme provided in A. B) For each condition, the amount of Luciferase that accumulated over time is provided after normalization to the activity measured 1 hour post infection. The graph presents AVG and SEM of 8 independent experiments.</p

PF74 induces increased Eu-repRNA availability to translation in purified HIV-1 cores <i>in vitro</i>.

<p>A) Cartoon presenting the experimental setup used to purify viral cores. Briefly, virion particles are passed by ultracentrifugation first through a 10% sucrose cushion containing detergent that deprives the virus of its membrane, and then through a 25% sucrose cushion through which viral cores pellet. Due to their fragility, viral cores are then either immediately analyzed by WB (B) or resuspended in rabbit reticulocyte lysate (RRL) in the presence of PF74 for 30 minutes prior to Luciferase measurement (C). The panel and the graph present data obtained from 3 independent experiments. As control for possible effects of PF74 on RRL activity, a control mRNA coding Luc (ctl mRNA) was used in parallel. *statistically significant differences following a Student t test (p≤0.05).</p

Effects of time dependent addition of high doses of PF74 on EURT and viral infectivity.

<p>A) Experimental scheme used here. Depending on the input virus (containing EU-repRNA, or a miniviral genome competent for a single round of infection and coding GFP), cells were either lysed at 10 hours post infection for EURT (B), or analyzed three days afterwards by flow cytometry (C). The graphs present averages and SEM of four independent experiments.</p