Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: SIV, SIV Δ env , SIV Δ nef , HIV-1 and HIV-1 Δ vpu were tested for particle release in the presence of human (hBST2) and rhesus (rBST2) tetherin. Virus production as a percentage of maximal particle release in the absence of tetherin is shown at increasing amounts of plasmid DNA for hBST2 (A) and rBST2 (B). The mean and standard deviation (error bars) for total p27 release is also shown for wild-type SIV, SIV Δ env and SIV Δ nef at the indicated amounts of DNA for hBST2 (C) and rBST2 (D). 293T cells were transfected in duplicate with proviral DNA constructs based on SIV mac 239 and HIV-1 NL4-3 together with expression constructs for either hBST2 or rBST2 (0, 2, 20 and 200 ng). Differences in the amount of plasmid DNA in each transfection were compensated by the addition of empty vector (pcDNA3). After 48 hours, the amount of virus released into the cell culture supernatant was measured by HIV-1 p24 and SIV p27 antigen-capture ELISA.

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Virus, Plasmid Preparation, Standard Deviation, Transfection, Construct, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: The accumulation of SIV p27 capsid in the cell culture supernatant relative to p55 Gag in cell lysates was compared for wild-type SIV, SIV Δenv and SIV Δ nef at increasing expression levels of human and rhesus tetherin. 293T cells were transfected with proviral DNA for wild-type SIV, SIV Δenv or SIV Δ nef together with 0, 2, 10 and 50 ng of plasmid DNA for hBST2 (A) or rBST2 (B). Forty-eight hours post-transfection, virus was recovered from the culture supernatant by centrifugation and cell lysates were prepared. Proteins were separated on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane and probed with monoclonal antibodies to p27/p55 Gag (183-H12-5C) and to BST2 (HM1.24). The blots were then probed with an HRP-conjugated goat anti-mouse secondary antibody, developed in chemiluminescent substrate and visualized using a Fujifilm Image Reader LAS 3000. Band intensities for p27 in virions and p55 in cell lysates were determined using the Image J Software (Rasband, W.S., Image, U.S. NIH, Bethesda, MD, http://rsb.info.nih.gov/ij , 1997–2008) and compared as p27/p55 ratios. ‘ND’ indicates that the p27 band was below the limit of detection.

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Cell Culture, Expressing, Transfection, Plasmid Preparation, Virus, Centrifugation, Membrane, Bioprocessing, Software

Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: HIV-1 Vpu and SIV Nef were tested for the ability to rescue virus release in trans for SIV Δ nef and a vpu -deficient strain of HIV-1 in the presence of human and rhesus tetherin. 293T cells were transfected in duplicate with 100 ng of proviral DNA for SIV Δ nef (A–D) or HIV-1 HXB2 (E–H), 50 ng of DNA for hBST2 or rBST2, and 100 ng of DNA for either HIV-1 Vpu, SIV Nef, SIV Nef G 2 A, SIV Nef crm − or HIV-1 Nef. G 2 A and crm − represent amino acid substitutions in the myristoylation site and the cholesterol recognition motif (L 129 R, Y 133 A and Y 134 A) of SIV Nef respectively. Vector controls included pcDNA3 for the tetherin expression constructs and pCGCG for the Nef expression constructs. Forty-eight hours post-transfection, the amount of virus released into the cell culture supernatant was measured by SIV p27 (A) or HIV-1 p24 (E) antigen-capture ELISA, and by infectivity on GHOST X4/R5 cells (C,G). To control for variability due to tetherin-independent effects of Nef and Vpu on particle release, the data are also expressed as percent maximal release in the absence of tetherin (B,D,F,H). The values above each bar represent the fold-increase in virus release compared to the empty vector control. The expression of wild-type SIV Nef, Nef G 2 A, Nef crm- , HIV-1 Nef and HIV-1 Vpu were verified by western blot analysis (I).

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Virus, Transfection, Plasmid Preparation, Expressing, Construct, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Control, Western Blot

Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: The ability of Nef to downmodulate tetherin from the cell surface was assessed by transfecting stable 293T cell lines expressing HA-tagged human or rhesus tetherin (hBST2 or rBST2) with bicistronic constructs expressing Nef and GFP. SIV Nef, SIV Nef G 2 A and SIV Nef crm − were expressed from the same mRNA transcript as GFP using pCGCG constructs in which expression of the GFP reporter gene was driven from an internal ribosomal entry site downstream of nef . Cells were also transfected with pCGCG without nef as an empty vector control. Twenty-four hours after transfection, the cells were stained with an anti-HA monoclonal antibody followed by an APC-conjugated, donkey anti-mouse polyclonal antibody and analyzed by flow cytometry. The values indicated in each plot represent the mean fluorescence intensity and standard deviation of HA/BST2 staining on GFP + cells for duplicate transfections.

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Control, Staining, Flow Cytometry, Fluorescence, Standard Deviation

Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: (A) Amino acid substitutions were introduced into full-length rBST2 and a deletion mutant lacking the first ten amino acids (rBST2 Δ10) at positions that differ from hBST2. The G 14 DIWK 18 motif of rBST2 was also introduced into hBST2 (hBST2 G 14 DIWK 18 ). Dashes represent sequence gaps, and positions that differ from wild-type rBST2 are indicated in red. (B) Expression of each of the rBST2 mutants tested in (C) was confirmed by western blot analysis of transfected 293T cell lysates. (C,D) SIV Nef was tested for the ability to rescue virus release for SIV Δnef in cells expressing each of the rBST2 and hBST2 mutants shown in (A). Transfection and assay conditions were the same as previously described.

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Mutagenesis, Sequencing, Expressing, Western Blot, Transfection, Virus

Journal: PLoS Pathogens

Article Title: Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

doi: 10.1371/journal.ppat.1000429

Figure Lengend Snippet: (A) Sequences corresponding to the cytoplasmic domain (N) and transmembrane domain (TM) of human and rhesus tetherin (hBST2 and rBST2) were exchanged to generate the chimeric fusion proteins rTM/hBST2, hTM/rBST2, rN/hBST2 and hN/rBST2. Amino acid sequences corresponding to the predicted transmembrane domains are boxed, dashes represent sequence gaps, and positions in hBST2 that differ from rBST2 are indicated in red. (B) The expression of each of these recombinant proteins was verified by western blot analysis. (C) Vpu was tested for the ability to rescue p24 release for HIV-1 HXB2 in 293T cells expressing hBST2, rBST2, rTM/hBST2, hTM/rBST2, rN/hBST2 and hN/rBST2. Transfection and assay conditions were the same as described previously.

Article Snippet: Transfections were performed in duplicate in 24-well plates seeded the day before at 5×10 4 cells per well using GenJet Lipid Transfection Reagents (SignaGen Laboratories, Gaithersburg, MD).

Techniques: Sequencing, Expressing, Recombinant, Western Blot, Transfection