Figure 1.
Design of Retrocyclin Constructs
(A) Shows a schematic of the four constructs (R1, R3, A1, and A3) used for stable transfections along with native retrocyclin cDNA. All constructs have two termination codons at the end to ensure read-fidelity (red). Constructs A1 and A3 contain additional downstream residues (orange), whereas constructs R1 and R3 lack them. The two arrows indicate the position at which the two site-directed mutagenesis (⊗17Q and R70K) were performed.
(B) Shows the three possible mature retrocyclin peptides that could be formed from the constructs, homodimers of R1 or A1 encoding RC-100 (wild-type retrocyclin), heterodimers of A1 and A3 or R1 and R3 encoding RC-101 (single lysine congener), and homodimers of R3 or A3 encoding RC-101_2K (double lysine congener).
Figure 2.
Extracts from HL60 Cells Stably Transfected with Retrocyclin Constructs Are Active against HIV-1 Infection
(A) TZM-bl cells were treated with extracts or peptide as indicated in the figure and infected with HIV-1 BaL (6.5 ng/ml p24) for 24 h. Infection was measured as percent luciferase activity compared to cells treated with control cell extract (average relative luciferase units [RLUs] of control HL60 extract = 178,200).
(B, C) PM1 cells and PBMCs were treated with extracts or peptide as indicated and infected with HIV-1 BaL (2 ng/ml p24) and cultured for 5–9 d. Bars represent percent BaL HIV-1 levels in the supernatants collected on days 5 (B) and 9 (C). The amount of p24 in PM1 cells treated with control extract = 76.85 ng/ml and in PBMCs treated with control extracts = 55.99 ng/ml.
(D) TZM-bl cells were treated with immunopurified (IP) extracts or peptides as indicated and infected with BaL HIV-1 (p24 = 2 ng/ml) for 24 h. Infection was quantified as percent luciferase activity compared to cells treated with control HL60 cell IP extracts (average RLU = 764,460). Error bars represent standard error of the mean (SEM). n = 3–4; #, p < 0.004; *, p < 0.002; **, p <0.0005.
(E) Cellular viability of TZM-bl cells treated with HL60 acid extracts as indicated was determined by measuring the reduction of MTT after 24h (n = 3). Bars represent percent viability as compared to vehicle control and error bars represent SEM.
(F) Cell death was monitored in PBMCs treated with the acid extracts by a trypan blue exclusion assay on day 9 (n = 1).
Figure 3.
Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides
(A) Retrocyclin peptides RC-100, RC-101, and RC-101_2K peptides (in duplicates) and (B) RC-100, RC-100b, RC-101, protegrin-1 (PG-1), rhesus theta defensin-1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3) were dotted (0–8 ng/4 μl dot) on a PVDF membrane and subjected to immuno-dotblot analysis.
(C) VC, R1R3, and A1A3 (100,000 cells each) were fixed onto glass slides and incubated with rabbit anti-RC-101 antibody followed by biotinylated goat anti-rabbit IgG secondary antibody and then stained using fluorescein isothiocyanate (FITC)-avidin. Slides were visualized using Zeiss Axiovert 200M microscope system at 40× magnification. The three rows show FITC staining, DIC, and the merged image, respectively. Scale bar represents 20 μm.
Figure 4.
Stably Transfected Promyelocytic Cells Produce Retrocyclin
(A) Shows the RP-HPLC trace of A1A3 cell extract (from 108 cells) and 50 μg of synthetic RC-101.
(B) Western blot of A1A3 HPLC fractions (23–28 min) using rabbit anti-RC-101 antibody. The arrows indicate the multimeric forms of retrocyclin observed in A1A3 fractions.
(C) TZM-bl cells were infected with HIV-1 (p24 = 2 ng/ml) in the presence or absence of pooled A1A3 fractions (final dilution 1:6 in D10) or 2 μg/ml of RC-101 for 24 h. Infection was quantified by luciferase measurement (average RLU of vehicle control with virus = 85,450).
Error bar represents SEM and n = 3–6; *, p < 0.0015; **, p <0.0001. MALDI-TOF MS spectra of Lys-C digested (D) synthetic RC-101_2K, (E) synthetic RC-101, and (F) A1A3 HPLC fraction 26 reveal that A1A3 cells produce RC-101.
Figure 5.
Aminoglycosides Mediate Read-Through of the Premature Termination Codon within the Retrocyclin Gene and Promote Anti-HIV-1 Activity
(A) Shows a schematic of the luciferase fusion constructs unrescued RC-101 and rescued RC-101 along with native retrocyclin cDNA.
(B) HOS-CD4-CCR5 cells cultured in antibiotic free medium (D10−) were transfected with unrescued RC-101 (negative control) or rescued RC-101 (positive control) plasmids along with phRL-CMV vector (transfection control). The next day transfected cells were treated with PBS for control cells or aminoglycosides at the indicated concentrations and allowed to grow for 24 h. Read-through was determined by measuring luciferase levels. Data are expressed as fold increase in luciferase expression normalized to renilla levels.
(C) TZM-bl cells grown in D10− were treated for 30 min with PBS, RC-101 (2.5 μg/ml), RC-100 (2.5 μg/ml), or aminoglycosides as shown in the figure and infected with HIV-1 BaL (2 ng/ml p24) for 24 h followed by luciferase measurement. Error bars represent SEM. n = 3–6; #, p < 0.007; * p < 0.0005; ** p <0.0001.
(D) TZM-bl cells cultured on cover slips were treated with PBS (Con) or 10 μg/ml tobramycin (Tob) and then immunostained with rabbit preimmune or antiretrocyclin serum using a biotinylated secondary antibody FITC-avidin system.
(E) Cellular cytotoxicity was assessed by performing an MTT assay on TZM-bl cells treated with indicated amount of peptide or aminoglycosides (n = 3). Bars represent percent metabolic inhibition as compared to control (vehicle + virus).
(F) TZM-bl cells, treated with either PBS, tobramycin (10 μg/ml), or RC-100 (2.5 μg/ml), were incubated with preimmune serum or antiretrocyclin serum as indicated and infected with HIV-1 (p24 of 5 ng/ml). Data are represented as percent infection. Error bars represent SEM. n = 3; +, p < 0.018 Statistical significance was determined by two-tailed Student's t-test.
Figure 6.
Expression of Retrocyclins in Cervicovaginal Tissue Model Using Aminoglycosides
(A) Cervicovaginal tissues were treated with PBS (Con) or 10 μl tobramycin (Tob) and incubated with rabbit preimmune serum or antiretrocyclin antibody. The slides were then incubated with biotinylated goat anti-rabbit IgG secondary antibody and then stained using FITC-avidin.
(B) Cytotoxicity was determined by measuring LDH activity in media underlying the tissues treated with PBS or tobramycin as indicated. Bars represent absorbance measured as 490 nm and error bars represent SEM; n = 6.
(C) HPLC trace of extracts of tissues treated with 10 μg/ml tobramycin (tissue + Tob) and 20 μg of synthetic RC-100.
(D) RC-100 synthetic peptide (indicated amounts), HPLC fractions 27–29 of control, tobramycin-treated, and RC-100 were dotted on a PVDF membrane and analyzed by immuno-dotblot.