PMC

Inhibition of B5 or C5 gp120-mediated cell-cell fusion by using PSC-RANTES or T-20. The 293T cells stably transformed with either pEXP-B5 gp120/HXB2 gp41 or pEXP-C5 gp120/HXB2 gp41 were added to CD4⁺/CCR5⁺ GHOST cells along with PSC-RANTES (1 or 100 nM), T-20 (1 or 100 nM), or no drug. HIV-1 Tat from the transfected 293T cells induces LTR-driven GFP expression in the Ghost cells upon cell fusion. (A) Decrease in GFP fluorescent/fused cells upon the addition of 1 or 100 nM PSC-RANTES. (B) Plot of relative GFP fluorescence in the presence compared to the absence of entry inhibitors.

Fitness of parental and env chimeric viruses relative to various primary HIV-1 isolates. (A) The parental and env chimeric B5 HIV-1 isolates were competed against C3, NL4-3_C5 gp120, C5, C8, C9, B4, and B6 primary HIV-1 isolates. The fitness difference (W_D) was calculated as the relative fitness value of B5 or NL4-3_B5 gp120 divided by the relative fitness value of the various competitor viruses. Relative fitness values were calculated by the formula, W = f₀/i₀, where f₀ is the production of individual HIV-1 isolates in a dual infection and i₀ is initial proportion of the respective isolate in the inoculum. (B) Same competitions as in panel A but with parental and env chimeric C5 HIV-1 isolates.

Use of yeast recombination/gap repair for cloning of divergent HIV-1 env genes. The gp120 coding regions of HIV-1 env genes from B5-91US056 and C5-97ZA003 share <82% nucleotide sequence identity (A) and differ at 11 of 46 amino acid positions within and flanking the V3 loop sequence (B). The entire HIV-1 gp120 coding sequence (aside from the leader sequence) was PCR amplified from primary B5 and C5 HIV-1 isolates and then transfected into yeast, along with the pREC-env(HXB2) vector. (C) Schematic illustration of the yeast recombination/gap repair cloning technique used to introduce diverse HIV-1 genes into expression (pEXP-env) or subcloning vectors (pREC-env) without the need or use of unique restriction enzyme sites. Details of the technique are provided by Marozsan and Arts ().

Kinetics of cell fusion mediated by transiently expressed B5 gp120/HXB2 gp41 or C5 gp120/HXB2 gp41. QT6 effector cells were cotransfected with pEXP-B5 gp120/HXB2 gp41 or pEXP-C5 gp120/HXB2 gp41 vectors and a β-lactamase expression construct containing T7 promoters and infected with a recombinant vaccinia virus encoding T7 polymerase (vTF1.1) to drive expression. These effector QT6 cells were then added to CD4⁺/CCR5⁺ HeLa (JC53) target cells labeled with CCF2-AM as previously described (, ). (A) Cell-cell fusion was detected by assaying for a shift from green to blue fluorescence, indicating β-lactamase cleavage of CCF2. Subpanels I, III, and V show the green fluorescence (405-nm excitation and 460-nm emission) of JC53 cells labeled CCF2-AM and not fused to effector cells, whereas panels II, IV, and VI show the blue fluorescence (405-nm excitation and 520-nm emission) resulting from the target cells fusing to the QT6 cells transfected with pcDNA3.1, pEXP-B5 gp120/HXB2 gp41 (B5), or pEXP-C5 gp120/HXB2 gp41 (C5), respectively. Fluorescent images were captured at 240 min. (B) After coincubation of the effector and target cells, the change in blue to green fluorescence was also monitored over a 240-min time course.

Direct time course competitions between parental and env chimeric primary HIV-1 isolates. Primary HIV-1 isolates, B5 and C5 or their chimeric counterparts, NL4-3_B5 gp120 and NL4-3_C5 gp120, were used in head-to-head competitions. Time course competitions in these PBMC were analyzed by using HTA at 8, 24, 48, and 120 h after dual infection (chimeric viruses in panel A). The heteroduplex representing relative production of the NL4-3_{B5 gp 120} and NL4-3_{C5 gp 120} chimeras in monoinfections are indicated in the right hand lanes and provide a reference for the heteroduplex migrations in the dual infections time course samples in the lefthand lanes. The relative B5 and C5 virus (or NL4-3_{B5 gp 120} and NL4-3_{C5 gp 120}) production derived from the HTA are plotted as a function of peak HIV-1 DNA production. In panels B and C, the solid line and closed circle represent the total HIV-1 DNA detected at each time point by real-time PCR relative to β-globin DNA, also amplified by real-time PCR. The relative B5 and C5 virus production at each time point was measured relative to the highest level of HIV-1 DNA copies detected for each time course. In panel B, ratios of B5 and C5 chimeric HIV-1 DNA (detected by HTA [panel A]) were plotted as relative to peak HIV-1 DNA production at 120 h.

Comparing relative fitness of subtype B and C HIV-1 isolates to gp120 expression on the virus particles. Pairwise competition experiments were performed with the primary B4, B5, B6, C3, C5, C8, and C9 HIV-1 isolates and the env chimeric viruses, NL4-3_B5 gp120 and NL4-3_C5 gp120. The actual competitions in the pairwise experiment are described in Materials and Methods and Results. (A) Total relative fitness values are plotted as the top gray bar and with the scale on the upper x axis. (B and C) Western blot analyses were performed on various dilutions (undiluted and 1;5, 1:25, and 1:125 dilutions of equal infectious virus units) of the primary B5, B6, C5, and C9 HIV-1, as well as the NL4-3_B5 gp120 and NL4-3_C5 gp120. Blots were probed with the B13 antibody specific for a conserved linear epitope in gp120. The intensities of the gp120 band on the Western blots were plotted relative to the intensity of B5 gp120 (black bars and scale on the bottom x axis in panel A). Blots of NL4-3_B5 gp120 and NL4-3_C5 gp120 were also stripped and probed with anti-gp41 and anti-p24 antibodies in panel B. In panel C, gp120, gp41, and p24 content was also measured in pelleted and sucrose cushion purified viruses of equal titer (NL4-3_B5 gp120 and NL4-3_C5 gp120).

Using CD4⁺/CCR5⁺ cells to measure the binding avidity of parental and env chimeric viruses. A competitive binding assay was performed by using the B5 and C5 parental and env chimeric viruses purified on sucrose cushions (Fig. ). Each of these viruses (MOI of 0.0001) were preincubated with CEM-NKR-CCR5 (CD4⁺/CCR5⁺/CXCR4⁺) or CEM-SS (CD4⁺/CXCR4⁺) cells and then competed off with either an NSI/R5 HIV-1 isolate (B2-92BR017) or an SI/X4 HIV-1 isolate (D1-92UG021) at increasing concentrations (MOI of 0.0001 to 0.0625). (A) Binding assay schematic diagram. (B) Binding of the NSI/R5 HIV-1 isolates or the SI/X4 strain (D1) to both CEM-NKR-CCR5 or CEM-SS cells at 15°C in the absence of competitor was measured by a quantitative reverse transcription-radioactive PCR assay. The viral RNA from each virus (sample virus and competitor) remaining on cells after washing was RT-PCR amplified and used in an HTA (see Materials and Methods). (C) HTAs performed on competitions involving B5, C5, NL4-3_B5 gp120, and NL4-3_C5 gp120 bound to CEM-NKR-CCR5 cells and then competed off with B2 virus. The lower HTA in panel C displays the inability of the SI/X4 D1 virus to compete off the NL4-3_B5 gp120 virus from CEM-NKR-CCR5 cells. The amounts of each parental or chimeric virus relative to the increased competitor virus (x axis) were plotted, and examples of NL4-3_B5 gp120 and NL4-3_C5 gp120 binding curves are shown in panel D. The binding constants (K_d), derived from each of these binding competitions curves (e.g., panel D) are shown in panel E.

Competitive fusion assays using cells stably expressing B5 gp120/HXB2 gp41 or C5 gp120/HXB2 gp41. Stable expression of B5 gp120/HXB2 gp41 or C5 gp120/HXB2 gp41 was measured in 293T cells transfected with the pEXP vectors and then passaged under zeocin selection. (A) Expression of the env glycoproteins in the 293T cells was measured by intracellular gp41 staining with anti-gp41 antibody (Chessie 8) and flow cytometry (see Materials and Methods). 293T cells expressing equal amounts of B5 gp120/HXB2 gp41 or C5 gp120/HXB2 gp41 (zeocin resistant) were then mixed with the target CD4⁺/CCR5⁺ U87 cells (puromycin/G418 resistant) in the presence or absence of selection media. (B) Relative B5 and C5 gp120-mediated fusion was monitored. (C) Effects of zeocin, puromycin, and G418 selection of 293T/B5 cells at 48 h and after a PBS wash. The U87.CD4.CCR5, 293T/B5, and 293T/C5 cells added alone or together to the selection media was then removed from the plates at 48 h and after the wash. Extracted DNA was also PCR amplified with mtDNA primers (see Materials and Methods). The relative intensities of the HIV-1 and mitochondrial PCR-amplified products are plotted in panel D. Panel E shows the relative B5 to C5 gp120-mediated 293T cell fusion to CD4⁺/CCR5⁺ U87 cells when different ratios of the two effector cells were added to each experiment. The relative amounts of the B5 to C5 env DNA were determined by using HTA and W_D determination as described in Materials and Methods.