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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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Blocking endogenous ps20 inhibits HIV-1 transfer. Jwt ps20inter or clone 3 (ps20inter) was treated with either a non-silencing (siNS) siRNA or a WFDC1/ps20-silencing siRNA for 6 days. ps20, GAPDH and HPRT mRNA was then measured by qRT-PCR relative to β-actin expression in either (A) Jurkat population or (B) Clone 3. Normalized relative expression was calculated in reference to siNS control. (C) 8 × 104 siRNA treated cells were dye-labelled and co-cultured with 40% 2044-infected donor Jurkat cells at a T:D ratio of 1:0.2. The mean percentage of Gag+ target cells in a 4 hour transfer assay is shown. (D) 2 × 105 Jwt cells and C3 clone from were pre-cultured for 3 days with 5 μg/ml of either control mouse IgG1 or the anti-ps20 Ab IG7, then washed, dye-labelled and co-cultured with 40% 2044-infected donor cells at a T:D ratio of 1:0.2 in the presence of a further addition of each Ab. Mean percentage of Gag+ ps20high target cells is shown after 4 hours of co-culture. (E) 2 × 105 Jwt cells and C3 clone cells were cultured in the absence (control, con) or presence of 1 ug/ml of rps20 for 16 hours, washed, dye-labelled and co-cultured with donor cells infected with 40% 2044 at a T:D cell ratio of 1:0.2. The percentage of Gag+ ps20 target cells is shown after 4 hours of co-culture. All data represent the mean of three replicate assays. Asterisk denotes statistically significant data as calculated using a paired t-test. *P ≤0.05; **P ≤0.01.
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Figure 4: Blocking endogenous ps20 inhibits HIV-1 transfer. Jwt ps20inter or clone 3 (ps20inter) was treated with either a non-silencing (siNS) siRNA or a WFDC1/ps20-silencing siRNA for 6 days. ps20, GAPDH and HPRT mRNA was then measured by qRT-PCR relative to β-actin expression in either (A) Jurkat population or (B) Clone 3. Normalized relative expression was calculated in reference to siNS control. (C) 8 × 104 siRNA treated cells were dye-labelled and co-cultured with 40% 2044-infected donor Jurkat cells at a T:D ratio of 1:0.2. The mean percentage of Gag+ target cells in a 4 hour transfer assay is shown. (D) 2 × 105 Jwt cells and C3 clone from were pre-cultured for 3 days with 5 μg/ml of either control mouse IgG1 or the anti-ps20 Ab IG7, then washed, dye-labelled and co-cultured with 40% 2044-infected donor cells at a T:D ratio of 1:0.2 in the presence of a further addition of each Ab. Mean percentage of Gag+ ps20high target cells is shown after 4 hours of co-culture. (E) 2 × 105 Jwt cells and C3 clone cells were cultured in the absence (control, con) or presence of 1 ug/ml of rps20 for 16 hours, washed, dye-labelled and co-cultured with donor cells infected with 40% 2044 at a T:D cell ratio of 1:0.2. The percentage of Gag+ ps20 target cells is shown after 4 hours of co-culture. All data represent the mean of three replicate assays. Asterisk denotes statistically significant data as calculated using a paired t-test. *P ≤0.05; **P ≤0.01.

Mentions: Extensive characterisation of the Dharmacon Accell siRNA showed a consistent 50-60% specific knockdown of ps20 mRNA with maximal effects seen in ps20inter populations. Accordingly, we conducted functional knockdown studies in the Jwt-ps20inter and clone 3 (ps20inter). Both populations were treated with either non-specific (NS) siRNA or siRNA against ps20, which inhibited ps20 mRNA significantly by 62% in the Jurkat population and by 54% in clone 3 (Figure 4A, B respectively). To control for off target effects, GAPDH and HPRT expression was also measured relative to β-actin and no significant modulation of either noted in the presence of the siRNA against ps20 (Figure 4A, B). A reduction in ps20 expression was associated with a significant 34% and 28% reduction in virus transfer into the WT Jurkat cells and clone 3, respectively (Figure 4C). These observations were supported by antibody-mediated blocking experiments. A significant 29% and 36% reduction in virus transfer into Jurkat and clone C3 respectively was noted when cultured with anti-ps20 Ab relative to control IgG (Figure 4D). Conversely, recombinant ps20 (rps20) promoted virus transfer. Cells were pre-cultured with 1 ug/ml rps20 over-night, generated as previously described [23], washed to remove excess protein, then co-co-cultured with infected targets, resulting in a significant 3.4-fold and 1.9-fold enhancement of virus transfer into Jurkat and clone C3 respectively (Figure 4E). Similar observations of Ab-mediated blockade and rps20-induced transfer were also noted in additional clones (data not shown). These data confirm that blocking endogenous ps20 in primary CD4+ T cells limits HIV-1 transfer.


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)

Blocking endogenous ps20 inhibits HIV-1 transfer. Jwt ps20inter or clone 3 (ps20inter) was treated with either a non-silencing (siNS) siRNA or a WFDC1/ps20-silencing siRNA for 6 days. ps20, GAPDH and HPRT mRNA was then measured by qRT-PCR relative to β-actin expression in either (A) Jurkat population or (B) Clone 3. Normalized relative expression was calculated in reference to siNS control. (C) 8 × 104 siRNA treated cells were dye-labelled and co-cultured with 40% 2044-infected donor Jurkat cells at a T:D ratio of 1:0.2. The mean percentage of Gag+ target cells in a 4 hour transfer assay is shown. (D) 2 × 105 Jwt cells and C3 clone from were pre-cultured for 3 days with 5 μg/ml of either control mouse IgG1 or the anti-ps20 Ab IG7, then washed, dye-labelled and co-cultured with 40% 2044-infected donor cells at a T:D ratio of 1:0.2 in the presence of a further addition of each Ab. Mean percentage of Gag+ ps20high target cells is shown after 4 hours of co-culture. (E) 2 × 105 Jwt cells and C3 clone cells were cultured in the absence (control, con) or presence of 1 ug/ml of rps20 for 16 hours, washed, dye-labelled and co-cultured with donor cells infected with 40% 2044 at a T:D cell ratio of 1:0.2. The percentage of Gag+ ps20 target cells is shown after 4 hours of co-culture. All data represent the mean of three replicate assays. Asterisk denotes statistically significant data as calculated using a paired t-test. *P ≤0.05; **P ≤0.01.
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Figure 4: Blocking endogenous ps20 inhibits HIV-1 transfer. Jwt ps20inter or clone 3 (ps20inter) was treated with either a non-silencing (siNS) siRNA or a WFDC1/ps20-silencing siRNA for 6 days. ps20, GAPDH and HPRT mRNA was then measured by qRT-PCR relative to β-actin expression in either (A) Jurkat population or (B) Clone 3. Normalized relative expression was calculated in reference to siNS control. (C) 8 × 104 siRNA treated cells were dye-labelled and co-cultured with 40% 2044-infected donor Jurkat cells at a T:D ratio of 1:0.2. The mean percentage of Gag+ target cells in a 4 hour transfer assay is shown. (D) 2 × 105 Jwt cells and C3 clone from were pre-cultured for 3 days with 5 μg/ml of either control mouse IgG1 or the anti-ps20 Ab IG7, then washed, dye-labelled and co-cultured with 40% 2044-infected donor cells at a T:D ratio of 1:0.2 in the presence of a further addition of each Ab. Mean percentage of Gag+ ps20high target cells is shown after 4 hours of co-culture. (E) 2 × 105 Jwt cells and C3 clone cells were cultured in the absence (control, con) or presence of 1 ug/ml of rps20 for 16 hours, washed, dye-labelled and co-cultured with donor cells infected with 40% 2044 at a T:D cell ratio of 1:0.2. The percentage of Gag+ ps20 target cells is shown after 4 hours of co-culture. All data represent the mean of three replicate assays. Asterisk denotes statistically significant data as calculated using a paired t-test. *P ≤0.05; **P ≤0.01.
Mentions: Extensive characterisation of the Dharmacon Accell siRNA showed a consistent 50-60% specific knockdown of ps20 mRNA with maximal effects seen in ps20inter populations. Accordingly, we conducted functional knockdown studies in the Jwt-ps20inter and clone 3 (ps20inter). Both populations were treated with either non-specific (NS) siRNA or siRNA against ps20, which inhibited ps20 mRNA significantly by 62% in the Jurkat population and by 54% in clone 3 (Figure 4A, B respectively). To control for off target effects, GAPDH and HPRT expression was also measured relative to β-actin and no significant modulation of either noted in the presence of the siRNA against ps20 (Figure 4A, B). A reduction in ps20 expression was associated with a significant 34% and 28% reduction in virus transfer into the WT Jurkat cells and clone 3, respectively (Figure 4C). These observations were supported by antibody-mediated blocking experiments. A significant 29% and 36% reduction in virus transfer into Jurkat and clone C3 respectively was noted when cultured with anti-ps20 Ab relative to control IgG (Figure 4D). Conversely, recombinant ps20 (rps20) promoted virus transfer. Cells were pre-cultured with 1 ug/ml rps20 over-night, generated as previously described [23], washed to remove excess protein, then co-co-cultured with infected targets, resulting in a significant 3.4-fold and 1.9-fold enhancement of virus transfer into Jurkat and clone C3 respectively (Figure 4E). Similar observations of Ab-mediated blockade and rps20-induced transfer were also noted in additional clones (data not shown). These data confirm that blocking endogenous ps20 in primary CD4+ T cells limits HIV-1 transfer.

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