Table 1.
Primer sets detecting gene expression of HIV-1 associated receptors.
Figure 1.
Basal HIV-1 receptor mRNA expression in resting epithelial cells.
TR146, FaDu, A431 and TZM-bl cells were examined for mRNA expression of CD4, CCR5, CXCR4, DC-SIGN and the HSPG's syndecan-1 and -4 by quantitative RT-PCR. Data are presented as mRNA transcripts (arbitrary units) normalized to β-actin in a minimum of three independent experiments. PBMCs showed significantly higher expression of CD4, CCR5 and CXCR4 than oral (TR146 and FaDu) or vaginal (A431) cell lines. A431 cells show significantly higher expression of SDC-1, whilst FaDu and A431 show significantly higher expression of SDC-4 than TR146. Bars indicate ± standard deviation from the mean. *** = P<0.001, * = P<0.05.
Figure 2.
Basal HIV-1 receptor surface expression in resting epithelial cells.
TR146, FaDu, A431, TZM-bl and NP2-R5 and –X4 expressing cells were examined for surface expression of CD4, CCR5, CXCR4, DC-SIGN, GalCer and HSPG's by flow cytometry using monoclonal primary antibodies specific to each receptor with a FITC-labeled secondary antibody. HSPG's were not analyzed in NP2-R5 or -X4 expressing cells. Data are presented as percentage of cells expressing each receptor in a minimum of three independent experiments. Bars indicate ± standard error of the mean. *** = P<0.001, ** = P<0.01, * = P<0.05.
Figure 3.
Different methods used to detect HIV-1 R5 and X4 binding to epithelial cells.
(A) Post-lysis detection of p24 gag protein by Western blotting. Primary (gingival) epithelial cells, TR146, FaDu, A431 and TZM-bl cells were incubated overnight (16–24 h) with cell free YU2 (R5) or LAI (X4). After extensive washing to remove unbound virus, normalised total protein lysates were separated by SDS-PAGE and probed for HIV p24 using α-actin as a loading control. (B) Detection of immobilized virus on the cell surface by flow cytometry. Epithelial cells were incubated overnight with cell free virus. Bound virus was detected using a Cy5-labeled anti-human secondary antibody to detect HIV-1 gp120 primary monoclonal on the APC channel. Electronic gates were set around an unlabelled cell control, this area is then set as zero and any cells shifted to the right of the gate are deemed positive. To determine amount of virus bound, virally exposed, labelled cell percentages are subtracted from the uninfected (unexposed) labelled control cell percentages to obtain the % fluorescence values shown. Data are representative of four independent experiments and bars indicate ± standard deviation from the mean. (C) Detection of packaged HIV R5 RNA by amplification of the HIV-1 pol gene using nested PCR. Total RNA was extracted from TR146, FaDu, A431 and TZM-bl cells incubated overnight with cell free YU2 (R5) and used to produce viral cDNA. This was then used as a template in a nested PCR to detect a 2 Kb region of HIV pol. (D) Percentage reduction in detection of immobilized virus on the cell surface by flow cytometry after trypsin treatment. Virally exposed cells are compared with cells labelled with secondary antibody alone. Data set is representative of three independent experiments. * = P<0.05.
Figure 4.
Post-integration HIV-1 mRNA transcription and de novo viral protein production in epithelial cells (MOI: 0.2).
(A) Detection of spliced HIV-1 tat mRNA in TR146, FaDu, A431 and TZM-bl control cells by PCR 24 h post-infection with YU2 (R5) or LAI (X4) infectious virus. Equal amounts of total RNA was used to synthesise viral cDNA which was then subjected to PCR using primers designed to span the TAT 1 and 2 exon junctions. (B) p55 gag protein detection in TR146, FaDu, A431 and TZM-bl control cells by Western blot after 24 h infection with R5 (YU2) and LAI (X4) virus. (C) Infection of TR146, FaDu, A431 and NP2-R5/X4 control cells with GFP-linked single-cycle X4, R5 and dual tropic HIV-1 gp160 pseudotyped virus and detection of GFP incorporation into epithelial cell DNA by flow cytometry. Error bars show standard error from the mean. Data are representative of three independent experiments.
Table 2.
Detection of integrated HIV-1 genome in epithelial cells by qPCR.
Figure 5.
HIV-1 entry via the endocytic pathway results in productive viral infection in epithelial cells.
Two fold serial dilutions of VSV-G pseudotyped HIV-1 (MOI 1 –0.125) were added to TR146, FaDu, A431 and TZM-bl control cells. Infection is measured 16–24 h later by flow cytometry as percentage of GFP expression. The effect of AZT (500 mM) on GFP expression was also measured at the highest virus inoculum. Error bars show standard error from the mean. Data are representative of three independent experiments
Figure 6.
Transfer of captured HIV-1 from epithelial cells to permissive cells via cell-cell contact.
TR146, FaDu and A431 cells were incubated with R5 (YU2) and LAI (X4) virus for 24 h and following extensive washing TZM-bl indicator cells were added for a further 48 h. Controls included FaDu, TR146 and A431 cells without the addition of virus. Data are representative of three independent experiments.
Figure 7.
Transfer of captured HIV-1 from epithelial cells to permissive cells via transcytosis.
TR146, FaDu and A431 were grown on polycarbonate transwell membranes and incubated with R5 (YU2) and LAI (X4) virus for 4 h. Following extensive washing the transwells were placed in a separate plate that overlaid a confluent monolayer of TZM-bl cells and incubated for a further 48 h at 37°C. TZM-bl cells were fixed, stained for β-galactosidase expression with X-Gal stain, and counted using light microscope at 100x magnification. Resulting replicate colony counts were averaged and analyzed by two factor (cell line and virus tropism) ANOVA and post hoc Fisher PLSD tests. Colony counts through A431 monolayers were significantly greater than those through TR146 and FaDu (P<0.05). Colony counts resulting from exposure of epithelium to X4 and R5 were significantly greater (P<0.05) than control wells with no virus exposure, but were not significantly different from each other. Data are representative of three independent experiments. * P<0.05.