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HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation.

St Gelais C, Coleman CM, Wang JH, Wu L - PLoS ONE (2012)

Bottom Line: HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells.However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells.Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4(+) T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+) T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+) T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+) T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.

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HIV-1 Nef enhances DC-mediated HIV-1 transmission to Hut/CCR5 T cells and modulates CD4 expression of DCs.Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Immature DCs were transduced with a Nef-expressing lentiviral vector (Nef+) or a negative control vector (Nef−). (A) Nef expression in vector-transduced DCs was detected at five days post-transduction by immunoblotting. HIV-1 p24 was used a positive control. (B and C) Nef expression in DCs promotes HIV-1 transmission. DC-mediated transfer of single-cycle, Nef-defective, R5-tropic luciferase HIV-1 to CD4+ Hut/CCR5 cells was measured at 2 days post-infection (dpi). The data show the mean ± SD of triplicate samples of cells from two different donors. (A–C) One representative experiment out of four is shown. cps, counts per second. (D and E) Transduced DCs were analyzed by flow cytometry for the positive percentage (D) and the mean intensity (E) of DC-SIGN, CD4 and CD86 expression at 5 days post-transduction. The data show the mean ± SD of three independent experiments. (B–E) *, Significant differences compared with the Nef-negative controls (P<0.05).
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pone-0034521-g001: HIV-1 Nef enhances DC-mediated HIV-1 transmission to Hut/CCR5 T cells and modulates CD4 expression of DCs.Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Immature DCs were transduced with a Nef-expressing lentiviral vector (Nef+) or a negative control vector (Nef−). (A) Nef expression in vector-transduced DCs was detected at five days post-transduction by immunoblotting. HIV-1 p24 was used a positive control. (B and C) Nef expression in DCs promotes HIV-1 transmission. DC-mediated transfer of single-cycle, Nef-defective, R5-tropic luciferase HIV-1 to CD4+ Hut/CCR5 cells was measured at 2 days post-infection (dpi). The data show the mean ± SD of triplicate samples of cells from two different donors. (A–C) One representative experiment out of four is shown. cps, counts per second. (D and E) Transduced DCs were analyzed by flow cytometry for the positive percentage (D) and the mean intensity (E) of DC-SIGN, CD4 and CD86 expression at 5 days post-transduction. The data show the mean ± SD of three independent experiments. (B–E) *, Significant differences compared with the Nef-negative controls (P<0.05).

Mentions: To examine whether Nef expression in DCs can modulate DC-mediated HIV-1 transmission to CD4+ T cells, immature monocyte-derived DCs (MDDCs) were transduced with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped, Nef-expressing lentiviral vector and then tested for the ability to transmit single-cycle HIV-1 to CD4+ Hut/CCR5 T cells. A VSV-G-pseudotyped Nef-deleted lentiviral vector was used as a negative control. These HIV-1-derived lentiviral vectors are defective for vif, vpr, vpu, and env genes [39]. Nef expression in the transduced MDDCs was confirmed by immunoblotting (Fig. 1A). The vector-transduced cells were pulsed with R5-tropic, Nef-defective, single-cycle luciferase HIV-1 [12], and co-cultured with CD4+ Hut/CCR5 T cells. Nef expression in DCs enhanced HIV-1 transmission by 6- to 8-fold (P<0.01) relative to the control vector-transduced DCs (Fig. 1B and 1C). These results indicate that Nef expression in DCs can promote DC-mediated HIV-1 transmission to CD4+ T cells.


HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation.

St Gelais C, Coleman CM, Wang JH, Wu L - PLoS ONE (2012)

HIV-1 Nef enhances DC-mediated HIV-1 transmission to Hut/CCR5 T cells and modulates CD4 expression of DCs.Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Immature DCs were transduced with a Nef-expressing lentiviral vector (Nef+) or a negative control vector (Nef−). (A) Nef expression in vector-transduced DCs was detected at five days post-transduction by immunoblotting. HIV-1 p24 was used a positive control. (B and C) Nef expression in DCs promotes HIV-1 transmission. DC-mediated transfer of single-cycle, Nef-defective, R5-tropic luciferase HIV-1 to CD4+ Hut/CCR5 cells was measured at 2 days post-infection (dpi). The data show the mean ± SD of triplicate samples of cells from two different donors. (A–C) One representative experiment out of four is shown. cps, counts per second. (D and E) Transduced DCs were analyzed by flow cytometry for the positive percentage (D) and the mean intensity (E) of DC-SIGN, CD4 and CD86 expression at 5 days post-transduction. The data show the mean ± SD of three independent experiments. (B–E) *, Significant differences compared with the Nef-negative controls (P<0.05).
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pone-0034521-g001: HIV-1 Nef enhances DC-mediated HIV-1 transmission to Hut/CCR5 T cells and modulates CD4 expression of DCs.Immature DCs were generated from purified monocytes by treatment with GM-CSF and IL-4 for 5 days. Immature DCs were transduced with a Nef-expressing lentiviral vector (Nef+) or a negative control vector (Nef−). (A) Nef expression in vector-transduced DCs was detected at five days post-transduction by immunoblotting. HIV-1 p24 was used a positive control. (B and C) Nef expression in DCs promotes HIV-1 transmission. DC-mediated transfer of single-cycle, Nef-defective, R5-tropic luciferase HIV-1 to CD4+ Hut/CCR5 cells was measured at 2 days post-infection (dpi). The data show the mean ± SD of triplicate samples of cells from two different donors. (A–C) One representative experiment out of four is shown. cps, counts per second. (D and E) Transduced DCs were analyzed by flow cytometry for the positive percentage (D) and the mean intensity (E) of DC-SIGN, CD4 and CD86 expression at 5 days post-transduction. The data show the mean ± SD of three independent experiments. (B–E) *, Significant differences compared with the Nef-negative controls (P<0.05).
Mentions: To examine whether Nef expression in DCs can modulate DC-mediated HIV-1 transmission to CD4+ T cells, immature monocyte-derived DCs (MDDCs) were transduced with a vesicular stomatitis virus G protein (VSV-G)-pseudotyped, Nef-expressing lentiviral vector and then tested for the ability to transmit single-cycle HIV-1 to CD4+ Hut/CCR5 T cells. A VSV-G-pseudotyped Nef-deleted lentiviral vector was used as a negative control. These HIV-1-derived lentiviral vectors are defective for vif, vpr, vpu, and env genes [39]. Nef expression in the transduced MDDCs was confirmed by immunoblotting (Fig. 1A). The vector-transduced cells were pulsed with R5-tropic, Nef-defective, single-cycle luciferase HIV-1 [12], and co-cultured with CD4+ Hut/CCR5 T cells. Nef expression in DCs enhanced HIV-1 transmission by 6- to 8-fold (P<0.01) relative to the control vector-transduced DCs (Fig. 1B and 1C). These results indicate that Nef expression in DCs can promote DC-mediated HIV-1 transmission to CD4+ T cells.

Bottom Line: HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells.However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells.Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4(+) T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+) T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+) T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+) T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.

Show MeSH
Related in: MedlinePlus