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WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

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HIV-1 transfer into J-ps20high cells is dependent on virus fusion and leads to higher levels of productive infection. (A) J-Ps20high and J-ps20inter target cells stained with DDAO SE vital dye were seeded at 1 × 105 cells per well of a 24 well plate in the presence or the absence of 5 μg/ml of T-20 for 1 hour prior to co-cultured with 18% Jwt ps20inter NL4-3-infected donor cells at a T:D ratio 1:0.2. Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells 4 hours post co-culture is shown. Data represent mean of three replicate assays. (B) The dye-labelled J-Ps20high and J-ps20inter target cells were seeded at 1 × 105 cells in the presence or the absence of 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 25% NL4-3-infected donor cells at a T:D ratio of (B) 1:0.2 or (C) 1:1. The percentage of Gag+ J-ps20inter vs. J-ps20high target cells +/- RT inhibitors were assessed at 4, 24 and 72 hours post co-culture. Data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using a paired t-test (*P ≤0.05; **P ≤0.01).
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Figure 2: HIV-1 transfer into J-ps20high cells is dependent on virus fusion and leads to higher levels of productive infection. (A) J-Ps20high and J-ps20inter target cells stained with DDAO SE vital dye were seeded at 1 × 105 cells per well of a 24 well plate in the presence or the absence of 5 μg/ml of T-20 for 1 hour prior to co-cultured with 18% Jwt ps20inter NL4-3-infected donor cells at a T:D ratio 1:0.2. Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells 4 hours post co-culture is shown. Data represent mean of three replicate assays. (B) The dye-labelled J-Ps20high and J-ps20inter target cells were seeded at 1 × 105 cells in the presence or the absence of 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 25% NL4-3-infected donor cells at a T:D ratio of (B) 1:0.2 or (C) 1:1. The percentage of Gag+ J-ps20inter vs. J-ps20high target cells +/- RT inhibitors were assessed at 4, 24 and 72 hours post co-culture. Data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using a paired t-test (*P ≤0.05; **P ≤0.01).

Mentions: Evidence exists for fusion -dependent and -independent T-T transfer of HIV-1 [6,36,37]. To probe this in the context of ps20, target cells were cultured with Jwt-ps20inter donor cells productively infected with NL4-3 at a T:D ratio of 1:0:2 for 4 hours in the presence or absence of the T-20 fusion inhibitor. T-20 addition reduced virus transfer significantly by 3-fold and 2.4-fold in the J-ps20inter vs. J-ps20high cells, respectively (Figure 2A). To determine productive infection [38], target cells were cultured with reverse transcription RT inhibitors prior to co-culturing with Jwt-ps20inter infected donor cells at a T:D ratio of 1:0.2 and Gag+ cells enumerated at 4, 24, and 72 hours post co-culture. J-ps20high target cells had higher infection with evidence of progressive increase in Gag+ cells from the 4 to 72 hour time point, whereas there was no significant virus spread in the J-ps20inter population (Figure 2B). The addition of RT inhibitors did not inhibit virus transfer in either population at 4 hours (Figure 2B). However, a significant reduction was observed in the J-ps20high population with a 1.6-fold and 3-fold reduction between the J-ps20high RT-inhibitor treated and untreated populations at 24 and 72 hours respectively (Figure 2B). RT-inhibitors have been noted not to influence HIV-1 transfer, but can inhibit Gag accumulation in prolonged co-cultures [37]. Our findings corroborate these observations. We next tested if increasing the virus challenge dose to 1:1 T:D ratio promoted virus spread in the J-ps20inter cells. Figure 2C shows increase of Gag+ cells at the 1:1 ratio from the 24-72 hour time point to be 1.83% (± 0.36) to 3.43% (± 0.78) respectively in J-ps20inter cells, versus 3.84% (± 0.45) to 9.34% (± 0.79) respectively in J-ps20high targets. At the lower T:D ratio, Gag+ staining increased from 1.82% (± 0.13) to 4.3% (± 0.28) in J-ps20high cells between 24-72 hours versus 0.66% (± 0.11) to 0.77% (± 0.05) in J-ps20inter cells (Figure 2B). These data confirm J-ps20inter cells require a higher virus challenge dose than J-ps20high for efficient virus spread to be achieved in these cells.


WFDC1 expression identifies memory CD4 T-lymphocytes rendered vulnerable to cell-cell HIV-1 transfer by promoting intercellular adhesive junctions.

Alvarez RA, Thorborn G, Reading JL, Reddy SK, Vyakarnam A - Retrovirology (2011)

HIV-1 transfer into J-ps20high cells is dependent on virus fusion and leads to higher levels of productive infection. (A) J-Ps20high and J-ps20inter target cells stained with DDAO SE vital dye were seeded at 1 × 105 cells per well of a 24 well plate in the presence or the absence of 5 μg/ml of T-20 for 1 hour prior to co-cultured with 18% Jwt ps20inter NL4-3-infected donor cells at a T:D ratio 1:0.2. Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells 4 hours post co-culture is shown. Data represent mean of three replicate assays. (B) The dye-labelled J-Ps20high and J-ps20inter target cells were seeded at 1 × 105 cells in the presence or the absence of 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 25% NL4-3-infected donor cells at a T:D ratio of (B) 1:0.2 or (C) 1:1. The percentage of Gag+ J-ps20inter vs. J-ps20high target cells +/- RT inhibitors were assessed at 4, 24 and 72 hours post co-culture. Data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using a paired t-test (*P ≤0.05; **P ≤0.01).
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Related In: Results  -  Collection

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Figure 2: HIV-1 transfer into J-ps20high cells is dependent on virus fusion and leads to higher levels of productive infection. (A) J-Ps20high and J-ps20inter target cells stained with DDAO SE vital dye were seeded at 1 × 105 cells per well of a 24 well plate in the presence or the absence of 5 μg/ml of T-20 for 1 hour prior to co-cultured with 18% Jwt ps20inter NL4-3-infected donor cells at a T:D ratio 1:0.2. Mean percentage of Gag+ J-ps20inter vs. J-ps20high target cells 4 hours post co-culture is shown. Data represent mean of three replicate assays. (B) The dye-labelled J-Ps20high and J-ps20inter target cells were seeded at 1 × 105 cells in the presence or the absence of 5 μM of RT-inhibitors (AZT+Lamimidine) for 1 hour prior to co-culture with 25% NL4-3-infected donor cells at a T:D ratio of (B) 1:0.2 or (C) 1:1. The percentage of Gag+ J-ps20inter vs. J-ps20high target cells +/- RT inhibitors were assessed at 4, 24 and 72 hours post co-culture. Data represent the mean of three replicate assays. Asterisks denotes statistically significant data as calculated using a paired t-test (*P ≤0.05; **P ≤0.01).
Mentions: Evidence exists for fusion -dependent and -independent T-T transfer of HIV-1 [6,36,37]. To probe this in the context of ps20, target cells were cultured with Jwt-ps20inter donor cells productively infected with NL4-3 at a T:D ratio of 1:0:2 for 4 hours in the presence or absence of the T-20 fusion inhibitor. T-20 addition reduced virus transfer significantly by 3-fold and 2.4-fold in the J-ps20inter vs. J-ps20high cells, respectively (Figure 2A). To determine productive infection [38], target cells were cultured with reverse transcription RT inhibitors prior to co-culturing with Jwt-ps20inter infected donor cells at a T:D ratio of 1:0.2 and Gag+ cells enumerated at 4, 24, and 72 hours post co-culture. J-ps20high target cells had higher infection with evidence of progressive increase in Gag+ cells from the 4 to 72 hour time point, whereas there was no significant virus spread in the J-ps20inter population (Figure 2B). The addition of RT inhibitors did not inhibit virus transfer in either population at 4 hours (Figure 2B). However, a significant reduction was observed in the J-ps20high population with a 1.6-fold and 3-fold reduction between the J-ps20high RT-inhibitor treated and untreated populations at 24 and 72 hours respectively (Figure 2B). RT-inhibitors have been noted not to influence HIV-1 transfer, but can inhibit Gag accumulation in prolonged co-cultures [37]. Our findings corroborate these observations. We next tested if increasing the virus challenge dose to 1:1 T:D ratio promoted virus spread in the J-ps20inter cells. Figure 2C shows increase of Gag+ cells at the 1:1 ratio from the 24-72 hour time point to be 1.83% (± 0.36) to 3.43% (± 0.78) respectively in J-ps20inter cells, versus 3.84% (± 0.45) to 9.34% (± 0.79) respectively in J-ps20high targets. At the lower T:D ratio, Gag+ staining increased from 1.82% (± 0.13) to 4.3% (± 0.28) in J-ps20high cells between 24-72 hours versus 0.66% (± 0.11) to 0.77% (± 0.05) in J-ps20inter cells (Figure 2B). These data confirm J-ps20inter cells require a higher virus challenge dose than J-ps20high for efficient virus spread to be achieved in these cells.

Bottom Line: Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer.Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, King's College London, UK.

ABSTRACT

Background: Elucidating mechanisms that promote HIV-1 transfer between CD4+ T-lymphocytes and their subsequent loss is of importance to HIV-1 pathogenesis. We recently reported that whey acidic protein, ps20, promotes cell-free HIV-1 spread through ICAM-1 modulation. Since ICAM-1 is pivotal in cell conjugation and intercellular HIV-1 transfer, this study examines ps20 effects on HIV-1 spread between T lymphocytes.

Results: We demonstrate intrinsic ps20 variability in primary CD4+ T-lymphocyte clonal populations and a significant positive correlation between endogenous ps20 levels and virus transfer involving fusion resulting in a spreading infection that could be reversed by the addition of reverse transcriptase inhibitors. Blocking anti-ps20 antibody or siRNA mediated ps20 knockdown, significantly reduced virus transfer. Conversely, virus transfer was promoted by ectopic ps20 expression or by exogenous addition of recombinant ps20. A higher frequency of virological synapse formation was evident in cocultures of HIV-1 infected donor T-cells with ps20high v ps20low/intermediate targets. Blocking ps20 inhibited T-lymphocyte conjugate formation and ICAM-1 expression, and was as potent as ICAM-1 in inhibiting HIV-1 transfer.

Conclusions: Therefore ps20 is a novel marker of CD4+ T-cells rendered vulnerable to HIV-1 infection by regulating the fundamental biologic process of intercellular conjugate formation and consequently of potential importance in HIV-1 pathogenesis.

Show MeSH
Related in: MedlinePlus