Figure 1.
The structure and sequence of HIV-1 Tat/TAR RNA.
A schematic of the secondary RNA structure of HIV-1 TAR is presented in panel (A) to describe the relative positioning/interaction with HIV-1 Tat, Cyclin T1, and CDK9. The sites of nucleotide sequence variability among HIV-1 isolates are indicated by the arrows. The bracketed letter or number associated with the polymorphism defines the HIV-1 subtype or circulating recombinant form, CRF (respectively). (B) The NMR structure of HIV-1 TAR complexed with a small molecule (RBT203) is shown in a space filling model (PDB file 1UUD in ball-and-stick). (C) Amino acid alignment of the consensus sequences of HIV-1 Tat proteins from all group M HIV-1 subtypes, which are responsible for the worldwide epidemic. (D) The consensus nucleotide sequence of the HIV-1 TAR RNA element in the various HIV-1 subtypes.
Figure 2.
Testing the susceptibility of HIV-1 to inhibition by Tat peptidomimetics.
(A) Comparison of the relative inhibition of the HIV-1 NL4-3 laboratory strain by three lead Tat peptidomimetics. Virus production in cell free supernatant was measured using an endogenous RT assay. (B) The concentration for 50% inhibition (IC50) of HIV-1 NL4-3 was calculated for the lead Tat peptidomimetics and for the reference antiretroviral, 3TC. (C) Cell penetration (hT4R5 fibroblasts) and nuclear localization of fluorescein-labeled L-51 peptide analyzed by confocal microscopy. (D) L-50 inhibition of viral replication of three primary CCR5-tropic HIV-1 isolates measured in U87.CD4.CCR5 cells (A1 is the subtype A Rawandan isolate A1-92RW009, B5 is the subtype B isolate B5-91US056 from the USA and C5 is the subtype C isolate C5-97ZA003 from South Africa) and of CXCR4-tropic strains (D1 is the Ugandan subtype D isolate D1-92UG021, E6 is the subtype A/E circulating recombinant isolate CRF01_AE from Thailand) as well as the laboratory strain HIV-1NL4-3 measured in U87.CD4.CXCR4 cell cultures. The IC50 values for L50 and 3TC were calculated from drug susceptibility curves (Figure S1). (E) Inhibition of HIV NL4-3 was also measure in human peripheral blood mononuclear cells stimulated with PHA/IL-2. The nucleoside RT inhibitor, 3TC or the non-nucleoside RT inhibitor, nevirapine (NVP) were used as controls. Virus production was measured at 8 and 10 days post-infection by the RT activity in the supernatant (cpm/mL).
Figure 3.
L50 inhibition of Tat-dependent transactivation in cell-free, reconstituted transcription system and in transfected cells.
DNA templates carrying the HIV promoter were transcribed in the presence or absence of recombinant Tat protein (+Tat) and increasing concentrations of the Tat peptidomimenic (L-50, left panel; L-51, right panel) (A); ρ identifies full-length transcripts and τ identifies transcripts ending at the terminator sequence inserted proximal to the promoter. Relative transcription levels of the Tat-dependent full-length transcript ρ decreased by 4-fold with the Tat peptidomimetic while the Tat-independent τ product remained almost unaffected. (B) 293T cells were transfected with the plasmids pLTR-luc (LTR) + Tat or pcDNA.LUC (CMV), where the luciferase gene was under the control of the HIV-1 LTR or CMV promoters, respectively. Cells were treated with either 2 or 10 µM L50. Relative light units, based on luciferase expression, were reported as percentages of the no drug controls. (C) L-50 was added to 293T cells transfected infectious molecular clone (pNL4-3) while expression of 293T cells transfected with luciferase reporter provided the control. Virus production from the 293T cells transfected with pNL4-3 was monitored by RT activity and expressed as a percent transcription.
Figure 4.
L50 effects on Tat-mediated transactivation of mRNA transcription in cell lines.
(A) A schematic of the dual transfection of pNL4-3 and pcDNA.LUC (CMV) or pLTR-luc in 293T cells. Luciferase (in lysates) (B) and RT activity (in cell-free supernatants) (C) was measured 72 h following transfection/drug treatment of 293T cells. Supernatant from 293T transfections conditions were used to infect U87.CD4.CXCR4 cells. (D) Virus production at 10 days post infection was measured by RT activity.
Figure 5.
Time-of-drug-addition experiment during synchronized HIV-1 infections.
Panel (A) provides a schematic representation of the experimental protocol, including the spinoculation step for synchronized virus infection and timing of drug additions over the 72 h time course. The relative time frame associated with each retroviral step is defined based on the time frame of sensitivity to the drug that blocks that particular replication step. Panel (B) plots the level of inhibition mediated by a specific drug added at a specific time post infection. Virus production, regardless of the timing of drug addition, was measured by luciferase activity at 72 h post infection. Curves are fitted to % inhibition mediated by the timed addition of each drug. Inhibition by a drug is absent after the completion of the specific step known to be a target of that drug. For example, AMD3100 bind CXCR4 and prevents HIV-1 entry; thus, inhibition of HIV-1 by AMD3100 is not observed if the drug is added >2 h post infection, i.e. after the HIV-1 entry step is complete. A biphasic curve was fitted to inhibition mediated by timed addition of L50. The first 12 hours of the timed drug addition was magnified to examine the inhibition of HIV-1 entry and reverse transcription (panel C; early events). These early steps of HIV-1 replication were removed from the plots in panel D (late events) to focus on the timed inhibition by integration and transcription inhibitors. The times of drug addition that maintains 50% inhibition (t1/2) are shown for each drug in the insets. All time-of-drug-inhibition experiments were performed in triplicate which resulted in a 10–15% variance in the inhibition levels. Error bars are not shown to prevent figure congestion.
Figure 6.
Time-of-drug-addition experiments during synchronized cell-to-cell fusion.
(A) A cell-to-cell fusion was mediated by an effector 293T cell, expressing HIV-1 Env gp120/gp41 (via pREC nfl transfection), binding to CD4 and CXCR4 receptors on a target U87 cell. The luciferase reporter is only expressed after HIV-1 Tat and Rev migrate from the effector cytoplasm to the target nucleus in the fused cell and transactivates/rescues LTR-driven mRNA expression. (B) The timing of 3TC, DRB, and L50 inhibition was measure in this synchronized cell-to-cell fusion where a smaller number of transfected 293T cells are pelleted onto adherent U87.CD4.CXCR4 cells. Luciferase expression is measure 48 h post cell fusion. The times of drug addition that maintains 50% inhibition (t1/2) are shown for each drug in the insets. Triplicate measurements resulted in a 10–15% variance in the inhibition levels.
Figure 7.
L50 effects on HIV-1 and MLV reverse transcription during cell culture infections.
(A) HIV-1 Luc-AM has the luciferase gene under the control of the SV40 promoter within the HIV-1 genome and in place of the deleted HIV-1 env gene. This virus, pseudotyped with exogenous HIV-1 Env expression, as used to infect U87.CD4.CXCR4 cells in the absence of drugs or in the presence of L-50, DRB, or 3TC. (B) MLV, harboring a GFP gene and pseudotyped with VSV-G Envelope glycoprotein, was used to infect 293T cells in the absence of drugs or in the presence of L-50 or AZT.
Figure 8.
Inhibition of minus strand strong stop DNA synthesis by L50.
(A) A reconstituted in vitro reverse transcription assay was performed with increasing L50 concentrations to determine the level of inhibition during (−) strand strong stop DNA synthesis. Quantitation using a phosphorimager was performed on full length (−) strand strong stop DNA products (full ssDNA; red box) as well as two intermediate paused DNA products (intermediate paused ssDNA; blue and green) and then plotted in panel (B). All experiments were performed in triplicate to calculate the L50 IC50 concentration (5.5±0.57 µM) to inhibit (−) strand DNA synthesis. IC50 values were identical for all three (−) ssDNA products.
Figure 9.
Minus strand strong stop DNA synthesis from wild type or mutant.
(A) Schematic representation of HIV-1 RNA fragments representing the R, U5, and primer binding sequence (PBS) in wild type (wt_R_u5_pbs), site-directed mutagenesis of the TAR bulge (mutant TAR), or truncated template lacking the majority of the R region. (B) Minus strand DNA products catalyzed by HIV-1 RT (p66/p51) and extended from an end labelled 18 nt primer binding to the pbs. The products were resolved on an 8% denaturing PAG. (C) Plot of the (−) strand strong-stop DNA product produced on the various RNA templates. Bands on the gel were quantified using a phosphorimager.