Journal: PLoS Pathogens

Article Title: Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

doi: 10.1371/journal.ppat.1004189

Figure Lengend Snippet: (A–B) Primary MDMs and autologous lectin/IL-2-activated CD4 + T cells were stimulated for 24 h with 544 U/ml (2 ng/ml) IFN-β eight days post isolation from buffy coats. (A) IFIT1 and Tetherin mRNA levels were determined by RT-qPCR and normalised to GAPDH. Bars represent the means ± standard deviations (SD) from four donors relative to untreated MDMs (−IFN, set at 1). (B) Tetherin protein levels were determined by western blot analysis of whole cell lysates containing equal amounts of total protein. Numbers above each lane indicate the Tetherin band intensities relative to untreated MDMs (−IFN, set at 1). (C–D) MDMs were stimulated for 24 h with 0–500 U/ml IFN-β eight days post isolation from buffy coats. (C) mRNA levels were determined as described for (A). Bars represent the means ± SD from three donors relative to untreated MDMs (set at 1). (D) Tetherin protein levels were determined by western blot analysis of whole cell lysates.

Article Snippet: Total RNA was isolated from cells using the RNeasy Plus Mini Kit (QIAGEN), and 50 ng of RNA reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. qPCR was performed as described above using 200 nM of IFIT1 and GAPDH primers from the IFNr qRT-Primer Set (Source BioScience), 200 nM of Bst-2 primers from the RT 2 qPCR Primer Assay (QIAGEN), and the following cycler programme: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, and 72°C for 10 min. No reverse transcriptase was added to control samples to confirm the complete elimination of genomic DNA.

Techniques: Isolation, Quantitative RT-PCR, Western Blot

Journal: PLoS Pathogens

Article Title: Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

doi: 10.1371/journal.ppat.1004189

Figure Lengend Snippet: (A) MDMs were infected with HIV-1 BaL for seven days, co-cultured with autologous CD4 + T cells for 2.5 h, fixed and immunostained for the indicated proteins. Arrows indicate VS. Scale bars = 10 µm. (B) BaL-infected MDMs were co-cultured with autologous T cells for the indicated times, fixed, and immunostained for p17 Gag and CD4. No T cells were added to a control sample of MDMs (0 min). The proportion of infected MDMs that associated with T cells (black+grey bars), and formed VS (black bars) was counted on seven random confocal images with a total of 146–208 infected MDMs for each time point. (C) BaL-infected MDMs were co-cultured with autologous T cells for the indicated times, the T cells were washed off the MDMs, and Gag DNA levels in the T cells were quantified by qPCR. As controls, T cells were incubated for 6 h with uninfected MDMs (uninf), or with infected MDMs in the presence of 500 nM NVP (6 h/NVP). (D) Autologous CD4 + T cells were incubated for 6 h with BaL-infected MDMs, or with cell-free supernatants collected from the same MDMs during the preceding 6 h period. All T cells were collected and Gag DNA levels in the T cells quantified by qPCR. (C–D) Gag DNA levels were normalised to GAPDH. For cell-cell transmission experiments, the levels of contaminating MDM-derived Gag and GAPDH DNA were subtracted from the total DNA levels. Graphs show the means ± SD of triplicate samples from a representative experiment relative to 6 h (set at 100%), or cell-free (set at 1).

Article Snippet: Total RNA was isolated from cells using the RNeasy Plus Mini Kit (QIAGEN), and 50 ng of RNA reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. qPCR was performed as described above using 200 nM of IFIT1 and GAPDH primers from the IFNr qRT-Primer Set (Source BioScience), 200 nM of Bst-2 primers from the RT 2 qPCR Primer Assay (QIAGEN), and the following cycler programme: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, and 72°C for 10 min. No reverse transcriptase was added to control samples to confirm the complete elimination of genomic DNA.

Techniques: Infection, Cell Culture, Incubation, Transmission Assay, Derivative Assay

Journal: PLoS Pathogens

Article Title: Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

doi: 10.1371/journal.ppat.1004189

Figure Lengend Snippet: (A) R3A-(+), -(−), or -Udel-infected MDMs, or uninfected control MDMs, were co-cultured with autologous CD4 + T cells for 6 h. As a control, 5 µM NVP was added to parallel MDM-T cell co-cultures. All T cells were separated from the MDMs, and aliquoted for qPCR and western blot analyses. For qPCR analysis, T cells were lysed immediately, and Gag DNA levels quantified by qPCR and normalised to GAPDH. The levels of contaminating MDM-derived Gag and GAPDH DNA were subtracted from the total DNA levels. Graphs for the “no NVP” samples show the means ± SD of triplicate samples from a representative experiment relative to R3A-(+) (set at 100%). Single values are shown for the “5 µM NVP” samples. For western blot analyses, T cells were lysed immediately (0 days), or after another two day-incubation (2 days). (B–C) T cells were co-cultured with R3A-infected MDMs for 6 h, separated from the MDMs, fixed, immunostained for p17 Gag and CD3, and analysed by flow cytometry. (B) shows the results of a representative experiment, and the numbers next to the gates indicate the proportions of cells with small (1×), medium (3×), and large (10×) p17 Gag clusters. (C) shows the mean proportions of cells with small (1×), medium (3×), and large (10×) p17 Gag clusters ± SD of duplicate samples from three donors, normalised to the MDM infection levels. Each donor is represented by differently shaped data points.

Article Snippet: Total RNA was isolated from cells using the RNeasy Plus Mini Kit (QIAGEN), and 50 ng of RNA reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. qPCR was performed as described above using 200 nM of IFIT1 and GAPDH primers from the IFNr qRT-Primer Set (Source BioScience), 200 nM of Bst-2 primers from the RT 2 qPCR Primer Assay (QIAGEN), and the following cycler programme: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, and 72°C for 10 min. No reverse transcriptase was added to control samples to confirm the complete elimination of genomic DNA.

Techniques: Infection, Cell Culture, Western Blot, Derivative Assay, Incubation, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Tetherin Can Restrict Cell-Free and Cell-Cell Transmission of HIV from Primary Macrophages to T Cells

doi: 10.1371/journal.ppat.1004189

Figure Lengend Snippet: (A) R3A-(+), -(−), or -Udel-infected MDMs, or uninfected control MDMs, were co-cultured with autologous CD4 + T cells for 6 h, the T cells separated from the MDMs and lysed immediately (0 days), or after another one to three day-incubation (1–3 days). Gag DNA levels in the T cells were quantified by qPCR and normalised to GAPDH. As a control, 500 nM NVP was added to parallel MDM-T cell co-cultures, and to the T cells after the co-culture [(+)+500 nM NVP]. The levels of contaminating MDM-derived Gag and GAPDH DNA were subtracted from the total DNA levels. Graphs show the means ± SD of triplicate samples from a representative experiment relative to R3A-(+) after three days (set at 100%). (B–C) T cells were co-cultured with R3A-infected MDMs for 6 h, separated from the MDMs and fixed immediately (0 days), or incubated for another five days (5 days). The T cells were immunostained for p24/p55 Gag and CD3, and analysed by flow cytometry. (B) shows the results of a representative experiment, and the numbers above the gates indicate the proportions of Gag-positive cells. (C) shows the mean proportions of Gag-positive cells ± SD from three donors.

Article Snippet: Total RNA was isolated from cells using the RNeasy Plus Mini Kit (QIAGEN), and 50 ng of RNA reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer's instructions. qPCR was performed as described above using 200 nM of IFIT1 and GAPDH primers from the IFNr qRT-Primer Set (Source BioScience), 200 nM of Bst-2 primers from the RT 2 qPCR Primer Assay (QIAGEN), and the following cycler programme: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, and 72°C for 10 min. No reverse transcriptase was added to control samples to confirm the complete elimination of genomic DNA.

Techniques: Infection, Cell Culture, Incubation, Co-Culture Assay, Derivative Assay, Flow Cytometry