CCR5-peptidoliposomes enhance the ability of soluble CD4 mimetic to inhibit infection of R5-tropic HIV-1.

<p>CCR5-peptidoliposomes were co-incubated for 2 h with R5-tropic JRFL-pseudotyped HIV-1 (A); R5-tropic ADA-pseudotyped HIV-1 (B); or X4-tropic HXB2-pseudotyped HIV-1 (C), in the presence or absence of different concentrations of sCD4M48. Peptide-free magnetic liposomes (in the absence of sCD4M48) were used as control (set to 100% infectivity). The virus was separated from the beads by a magnetic field and TZM-HeLa-β-gal target cells were infected for 4 h. β-gal expression was carried out 48 h post infection. An unpaired <i>t</i>-test (two-tailed) was used to assess the significance of the difference in the means observed between the two groups indicated, <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

CCR5-peptidoliposomes bind soluble recombinant gp120.

<p>Peptidoliposomes were incubated with 4 μM soluble recombinant His-tagged gp120 for 1 h at 37°C and the binding was assessed as described in the Materials and Methods. Mimetics used: mCD4 –CD4M48-PAL (1%); mCCR5-Nt – NT-2Y-CCR5-PAL (1%); mCCR5-ECL2 –ECL2-CCR5-2PAL (1%); mCCR5-Nt-3FSN–NT-3FSN-CCR5-PAL (1%); sCD4M48 –soluble M48 peptide (10 μM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref015" target="_blank">15</a>]. Control – peptide-free magnetic liposomes. <i>p</i> < 0.05. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

Magnetic liposome population is homogeneous.

<p>The magnetic liposomes were prepared using a lipid mixture POPC:POPE:DMPA (molar ratio 6:3:1) containing 1% Biotinyl-DOPE in the presence (black peak) or absence (white peak) of 1% Rhodamine–DOPE, and analyzed by FACS as described in the Materials and Methods. FL1-H designates the height of the photon peak obtained by using a 525/50 band pass filter (FL1). The figure shows the data for an experiment representative of two similar experiments.</p

Palmitoylated CCR5-peptidomimetics can be displayed on liposome surface.

<p>Peptidoliposomes were generated in the presence of increasing concentrations (molar %) of NT-2Y-CCR5-PAL (A) or ECL2-CCR5-2PAL (B) in the lipid mixture. (C) Peptidoliposomes were formed in the presence of 1% ECL2-CCR5-2PAL and the indicated concentrations of NT-2Y-CCR5-PAL in the lipid mixture. Peptidoliposomes were reacted with anti-CCR5 N-terminus polyclonal antibody (ab 7346, Abcam) (A) or with anti-CCR5 ECL2 (raised against a synthetic peptide 2D7-2SK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144043#pone.0144043.ref044" target="_blank">44</a>]) polyclonal antibody (ab 36818, Abcam) (B and C), followed by a secondary HRP-conjugated antibody (ab 7090, Abcam) and analyzed as described in the Materials and Methods. Control – peptide-free magnetic liposomes. The data are mean ± S.E.M. calculated from three independent experiments each performed in triplicate.</p

HIV-1 incubation with CCR5-peptidoliposomes does not deplete viral loads.

<p>JRFL-pseudotyped HIV-1 was incubated for 2 h with peptidoliposomes in the presence or absence of M48 peptide–sCD4M48 (10 μM). CCR5-Beads - peptidoliposomes containing 5% each of NT-3FSN-CCR5-PAL and ECL2-CCR5-2PAL; (CD4+CCR5)-Beads - peptidoliposomes containing 5% each of CD4M48-PAL, ECL2-CCR5-2PAL and NT-3FSN-CCR5-PAL. At the end of the incubation, the supernatant and liposome fractions were analyzed by ELISA for the presence of HIV-p24 antigen as described in the Materials and Methods. Peptide-free magnetic liposomes were used as the control (p24 count in the supernatant set to 100%). The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p

Effect of co-display of CD4- and CCR5-peptidomimetics on the ability of CCR5-peptidoliposomes to inhibit HIV-1.

<p>R5-tropic JRFL-pseudotyped HIV-1 was co-incubated for 2 h with: CCR5-Beads–peptidoliposomes containing 5% each of NT-3FSN-CCR5-PAL and ECL2-CCR5-2PAL, or (CD4+CCR5)-Beads–peptidoliposomes containing 5% each of CD4M48-PAL, ECL2-CCR5-2PAL and NT-3FSN-CCR5-PAL. Peptide-free magnetic liposomes were used as control (set to 100% infectivity). The virus was separated from the beads by a magnetic field and TZM-HeLa-β-gal target cells were infected for 4 h. β-gal expression was carried out 48 h post infection. The data are mean ± S.E.M. calculated from three independent experiments each performed in duplicate.</p