The resistance of an R88-A3G-transduced CD4+ C8166 T cell line against HIV-1 infection.

<p>A). Equal amounts of pNL4.3-GFP virus were used to infect vector-, HA-A3G-, or R88-A3G-transduced C8166 cell lines. After 48 hours of infection, virus-containing supernatants (passage 1) were collected, and the same volume of infectious supernatant was used to infect fresh and transduced C8166 cells (passage 2). The same procedure was performed again until virus-containing supernatants at assage 3 were obtained. Then, levels of HIV-1 Gag-p24 antigen in supernatants from passages 1, 2, and 3 were measured by HIV p24 ELISA assay. B). To test the effect of R88-A3G on virus infectivity, equal amounts (adjusted by Gag-p24 level) of virus from passage 1 were used to infect fresh R88-A3G-transduced C8166 cells. At different time points, the supernatants were collected and the HIV Gag-p24 level was measured to monitor virus replication. Also, at day 6 post-infection, the percentage of HIV-infected (GFP-positive) cells was evaluated by the flow cytometry assay (C).</p

Expression of R88-A3G does not affect virus maturation, but significantly inhibits viral cDNA synthesis.

<p>A). R88-A3G does not affect virus maturation. HxBru-Vif⁻ and Vif⁺ viruses produced from 293T cells co-transfected with corresponding provirus and HA-A3G or R88-A3G expressor were pelleted, lysed and processed for immunoprecipitation with human anti-HIV serum. The immunoprecipitates were submitted to 10% SDS-PAGE and analyzed by Western blotting using rabbit anti-anti-RT antibody (upper panel) and anti-p24 antibodies (lower panel). B). R88-A3G inhibits viral cDNA synthesis at early stage of viral infection. Dividing C8166 T cells were infected with equal amounts of different HxBru-Vif⁺ virus stocks, which were produced from 293T cells co-transfected with the provirus and HA-A3G or R88-A3G expressor. At 12 hours post-infection, 1×10⁶ cells were lysed and the total viral DNA was detected by PCR using HIV-1 LTR-Gag primers, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001995#s4" target="_blank">Materials and Methods</a>. In parallel, the content of cellular β-globin DNA in each sample were also examined by specific PCR analysis (lower panel).</p

The R88-A3G is stably expressed in CD4+ C8166 cells and its presence does not affect cell growth and CD4 receptor expression.

<p>C8166 T cells (10×10⁶) were transduced with lentiviral vectors containing R88-A3G (pYEF1-R88-A3G-puro), HA-A3G (pYEF1-HA-A3G-puro) transgenes or empty vector (pYEF1-MCS-Puro), and puromycin-resistant cell population was selected by puromycin (0.5 µg/ml). Once the puromycin-resistant cell population was obtained, different analyses were performedfor their characterization. A). To detect R88-A3G and HA-A3G expression in C8166 T cells, transduced cells were infected with HxBru-Vif⁻ and HxBru-Vif⁺ virus. After 72 hours of infection, the produced viruses in the supernatant were concentrated by ultracentrifugation over 20% sucrose cushions. Then, virus pellets were lysed and resolved by 10% SDS-PAGE followed by Western blotting using anti-A3G and anti-p24 antibodies. B). The CD4 receptor expression levels observed on vector- and R88-A3G-transduced C8166 T cells were analyzed by using anti-CD4 staining and a flow cytometry assay. C). The cell cycle profile of vector- and R88-A3G-transduced C8166 T cells was analyzed by measurement of the cellular DNA content by staining with 30 µg/ml of propidium iodide (PI) and flow cytometry assay. D). To assess the growth of vector- and R88-A3G-transduced C8166 T cells, a WST assay was performed to determine cell viability at different time points. Each experiment was performed in triplicate and repeated at least three times. Results are shown as the mean ± SD of representative experiments.</p

Expression of R88-A3G in HIV-1 producing cells inhibits virus infectivity in the presence of Vif.

<p>Viruses were produced from 293T cells transfected with 3 µg of HIV-1 HxBru-Vif⁺ or HxBru-Vif⁻ with 4 µg of HA-A3G or R-A3G expressor. Then, equal amounts of produced viruses (as adjusted by the Gag-p24 level) were used to infect HeLa-CD4-CCR5-β-Gal cells and CD4+ C8166 T cells. A). At 48 hours post-infection, HIV-1 infected cells were detected by MAGI assay (left panel) and counted by Elispot Reader (right panel). B). At 48 hours post-infection, virus production in of C8166 cells were monitored by measurement of HIV-1 Gag-p24^gag antigen in the supernatants with p24 ELISA assay (upper panel). Cell-associated HIV-1 Gag-p24 (middle panel) and Vif (lower panel) were detected by Western blotting with anti-p24 or anti-Vif antibodies, respectively. C). pNL4.3-GFP viruses were first produced from 293T cells co-transfected by pNL4.3/GFP provirus with HA-A3G or R88-A3G. Then, equal amounts of pNL4.3-GFP viruses were used to infect C8166 T cells. At 72 hours post-infection, the percentage of infected (GFP-positive) cells was measured by FACS analysis. D). Dose-dependent effect of R88-A3G on HIV-1 infectivity. Different amounts of R88-A3G plasmid (0, 0.2, 1, 2, 4 µg) and pNL4.3-GFP proviral DNA (3 µg) were used to co-transfect 293T cells. Viruses from each transfected cell culture were collected and equal Gag-p24 amounts of viruses were used to infect C8166 T cells. 72 hours later, percentage of infected (GFP-positive) cells was measured by FACS analysis (left panel). Meanwhile, the expression of R88-A3G in the corresponding transfected 293T cells was detected by Western blotting with anti-A3G antibody (right panel).</p

HIV-1 virus generated from CD4+ T cells stably expressing R88-A3G lost infectivity in human primary blood mononuclear cells (hPBMCs).

<p>Equal amounts of pNL4.3-GFP viruses produced from vector- or R88-A3G-transduced C8166 cells (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001995#pone-0001995-g006" target="_blank">figure 6</a>, passage 1) were used to infect 1×10⁶ PHA-stimulated human PBMCs for 8 hours. After infection, PBMCs were washed and at different time intervals, virion-associated Gag-p24 antigen levels in the supernatant were measured by anti-p24 ELISA (A). Values were representative of data obtained from two independent experiments. At 5 days post-infection, the numbers of infected (GFP-positive) cells in mock-infected, or differently HIV-1 infected hPBMC cells, as indicated, were visualized under fluorescence microscopy (B).</p

R88-A3G is sensitive to Vif-induced degradation, but is efficiently incorporated into Vif⁺ HIV-1 particles.

<p>A). Effect of Vif on the degradation of R88-A3G. HA-A3G or R88-A3G expressor was transfected alone or co-transfected with pcDNA-hVif expressor in 293T cells. At 48 hour post-transfection, cells were pulse radiolabeled with [₃₅S]-methionine for 30 min, and labeled cells were collected and lysed at 0, 1.5 and 5 hours after pulse-labeling. Then, the level of HA-A3G in each lysed-cell sample was detected by anti-HA immunoprecipitation (upper panel). The level of R88-A3G in each lysed-cell sample was evaluated by anti-Vpr immunoprecipitation (middle panel). To detect HIV-1 Vif expression in different transfected cell samples, an aliquot of transfected cells from each culture was lysed and directly loaded onto 12.5% SDS-PAGE followed by Western blotting with anti-Vif antibody (lower panel). B). R88-A3G was efficiently incorporated into viral particles in the presence of Vif. 293T cells were co-transfected with HxBru-Vif+ (3ug) and HA-A3G or R88-A3G expressor (2ug). After 48 hours cells were collected and the produced virus particles were collected from supernatant by ultracentrifugation through a 20% sucrose cushion. Both cell and virus lysate samples were directly loaded onto a 12% SDS-PAGE gel and analyzed by Western blotting with rabbit anti-A3G and anti-p24, as indicated (upper panel). The ratio of R88-A3G or HA-A3G incorporation into the viral particle relative to the total amount of R88-A3G or HA-A3G was also quantified by laser densitometry (lower panel). The data presented herein are the means and standard deviations from two independent experiments.</p

R88-A3G was expressed in viral producing cells and cleaved by viral protease.

<p>A). Schematic representation of constructs of HA-A3G and R88-A3G. HA-tag or Vpr14-88 was fused in frame to the N-terminus of Apobec3G (A3G). An HIV-1 protease cleavage site (PCS) was inserted between R88 and A3G. B). HA-A3G or R88-A3G was expressed in 293T cells. 293T cells were transfected with the HA-A3G or R88-A3G expressor. After 48 hours, cell lysates were immunoprecipitated with a mixture of anti-HA and anti-Vpr antibodies followed by Western blotting with anti-A3G antibody. C). Cleavage of R88-A3G in virus produced from HIV-1 expressing cells. 293T cells were cotransfected with HxBru-Vif⁻ provirus and the HA-A3G or R-A3G expressing plasmid. After 48 hours, produced viruses were pelleted from supernatant by ultracentrifugation through a 20% sucrose cushion, lysed, and directly loaded onto 10% SDS-PAGE followed by Western blotting with anti-A3G polyclonal antibody (upper panel) or with anti-p24 monoclonal antibody (lower panel). The positions of R88-A3G and the cleaved product of A3G are indicated at right side of gel.</p

Comparison of prevalence, risk factors and outcomes of cataract surgery from selected population-based studies in Asia.

<p>APEDS = Andhra Pradesh Eye Disease Study; SiMES = Singapore Malay Eye Study; SINDI = Singapore Indian Eye Study; ACES = Aravind Comprehensive Eye Study; CIEMS = Central India Eye & Medical Study; BCVA = best corrected visual acuity; PCO = posterior capsular opacification; CME = cystoid macular edema; AMD = age-related macular degeneration; DR = diabetic retinopathy.</p><p>“−”: not reported.</p>*<p>Age-standardized to the Indian adult population from the 2010 Singapore Census.</p

Socioeconomic and systemic factors associated with post-operative visual impairment in the 1^st and 2^nd or higher generation Indian migrants living in Singapore.

<p>VI = visual impairment, defined as presenting visual acuity ≤20/60. OR = odds ratio; CI = confidence interval.</p>*<p>p<0.05.</p

Causes of post-operative visual impairment in the 1^st and 2^nd or higher generation Indian migrants living in Singapore.

<p>PVA = presenting visual acuity; BCVA = best-corrected visual acuity.</p><p>“Other” included one individual with pterygium, one with phthisis, one with trauma and one with myopic maculopathy. The exact cause in three individuals cannot be determined.</p