Interestingly, an IFN-induced loss of particle infectivity of newly synthesized HIV-1 particles has been described in PHA/IL-2-stimulated PBMCs [38] and myeloid U937 cells [39]. the ratio obtained for TZM-bl cell lysates (B) or the ratio obtained for untreated macrophages (C) was set to 1 1, respectively. 1742-4690-10-111-S1.pdf (435K) GUID:?91DA77D4-D7B5-4A19-A178-16EAC3D9F4A0 Additional file 2: Figure S2 90K reduces the particle infectivity of multiple HIV-1 strains. (A) 293T cells were cotransfected with the indicated proviral plasmids and highest amount of pcDNA6.90K-myc or empty vector. Supernatants were analyzed for infectious HIV-1 using a luminometric TZM-based luciferase assay. Shown are the results of one representative experiment out of three-six. (B) Relative levels of particle infectivity, defined as HIV-1 infectivity per ng p24 capsid are depicted. (C) Sucrose cushion-purified virions were analyzed by immunoblotting. Percentages indicate the relative gp120 incorporation, as measured by Infrared imaging-based quantification of the amount of gp120 per p24. The signal intensity in absence of 90K expression was set to 100%. (D) Cell lysates were analyzed by immunoblotting using the indicated antibodies. Numbers indicate the efficiency of gp160 processing. * : p? ?0.05; **: p? ?0.02 (Students T-Test). 1742-4690-10-111-S2.pdf (500K) GUID:?36D643B2-FD20-4FDA-9095-763C2E8FE80D Additional file 3: Figure S3 90K-myc expression is not associated with FRP toxicity or reduced metabolic activity. (A) 293T cells were transfected with pcDNA6.90K-myc (1.3?g), empty vector, or UV-irradiated and stained two days post transfection and one day post UV-irradiation with 7-AAD. Shown are representative dot plots of one experiment out of two. Numbers indicate percentage of 7-AAD-positive cells. (B) Quantification of 7-AAD FACS analysis. (C) Cells were lysed and analysed for metabolic activity by Cell Titer Glow assay. Shown are the RLU of triplicates obtained from one representative experiment out of two. 1742-4690-10-111-S3.pdf (415K) GUID:?FF31A2B0-A3AA-470F-ACA4-D5FCE3E82BCD Additional file 4: Figure S4 90K does not reduce the cell surface levels of CD4. (A) 293T cells were cotransfected with pcDNA.CD4 and pIRES2EGFP.90K-myc or empty vector, Cells were stained with APC-conjugated anti-CD4 and analyzed by flow cytometry. Shown are representative dot plots of one experiment out of three. (B) 293T cells were cotransfected with pcDNA.CD4 and pVpu-IRES GFP or empty vector and processed like in (A). (C) CD4 cell surface levels were calculated by comparing, within the same sample, CD4 levels on non-GFP-expressing cells (gate P2) with CD4 levels on cells with medium-high GFP expression levels 10Panx (gate P3). CD4 levels on vector transfected cells were set to 100%. (D) An aliquot of the cells shown in (A) and (B) were lyzed and analyzed by Western 10Panx Blotting using the indicated antibodies. 1742-4690-10-111-S4.pdf (396K) GUID:?94979091-35F0-4216-B527-EA0BBFB2D9DD Additional file 5: Figure S5 90K and Env colocalize to a high extent. (A) 293T cells were cotransfected with pcDNA6.90K-myc and an HIV-1 10Panx Env expression plasmid, and stained for 90K-myc (green) and Env (red). Scale bar: 10?m. (B) The classic colocalization coefficient was calculated for the colocalization of 90K protein with Env protein or using ZEN2010 software. The data represent the arithmetic mean S.D. of 105 analyzed cells. 1742-4690-10-111-S5.pdf (884K) GUID:?A3866E6C-78B3-4FAB-ACF9-3E6564246449 Additional file 6: Figure S6 No evidence for a direct interaction of 90K and HIV-1 Env. (A-C) 293T cells were cotransfected with pcDNA6, pcDNA6.90K-myc, an HIV-1 Env expression plasmid, pcDNA.CD4 or a combination out of these. (A) 90K, CD4 and bound proteins were precipitated from cell lysates by an anti-90K or anti-CD4 antibody, respectively. (B) 90K, CD4 and bound proteins were precipitated from cell lysates by an anti-myc or anti-CD4 antibody, respectively. (C) Env and bound proteins were precipitated from cell lysates by an anti-gp120 antibody. For each experimental set up, an aliquot of whole cell lysate for expression control (Input) and the precipitated proteins were analyzed by Immunoblot with indicated antibodies. 1742-4690-10-111-S6.pdf (536K) GUID:?9C66CC7F-58D3-46E1-8C4C-CC2B8726F4BA Additional file 7: Figure S7 90K does not retain Env in the ER. (A-B) 293T cells were cotransfected with pBR.HIV-1 IRES 10Panx GFP and vector or pcDNA6.90K-myc. (A) Cell lysates were treated with PNGase. (B) Cell lysates were treated with EndoH. Deglycosylated and control proteins were analyzed by Western Blot. Numbers indicate the efficiency of gp41 processing, calculated as the signal ratio of gp41 relative to (gp41?+?gp160), or the percentage of deglycosylated gp160 to the total gp160 signal. 1742-4690-10-111-S7.pdf (423K) GUID:?26BD5A38-24A0-463A-8F50-F0597B6A67A8 Additional file 8: Figure S8 Species specificity of 90K-imposed antiviral activity. (A) 293T cells were cotransfected with pBR.HIV-1 NL4.3-IRES GFP and 1.3?g of the indicated expression constructs or empty vector. Two days post transfection, supernatants were analyzed for particle infectivity, defined as infectivity per ng p24. Cell lysates were analyzed by immunoblotting. (B) Sucrose cushion-purified virions were analyzed by immunoblotting. Shown is the relative gp120 incorporation, as measured by Infrared imaging-based quantification of the amount of gp120 per p24. The signal intensity in absence of 90K expression was set to 100%. (C) Cell lysates were analyzed by.