7 research outputs found

    Nef alleles of SIV<sub>smm/mac</sub>, HIV-2 and HIV-1 counteract rhesus macaque and sooty mangabey tetherin, but not human tetherin.

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    <p>Nef alleles of SIV<sub>smm/mac</sub>, HIV-2 and HIV-1 were tested for the ability to rescue particle release for SIV <i>Δnef</i> in the presence of human tetherin (hBST2), rhesus macaque tetherin (rBST2) and sooty mangabey tetherin (sBST2). (A) The amino acid sequences corresponding to the cytoplasmic domains of hBST2, rBST2 and sBST2 are shown. Dashes represent sequence gaps and residues that differ from rBST2 are indicated in red. The mean and standard deviation (error bars) for total p27 release (B) and for percent maximal release (C) are shown for the indicated Nef alleles of SIV<sub>smm/mac</sub>, HIV-2 and HIV-1 in the presence of hBST2, rBST2 and sBST2. (D) Protein expression was confirmed for SIV p55 Gag, BST2, HIV-1 Vpu and for each of the Nef alleles by western blot analysis of 293T cell lysates. The Nef proteins of SIV<sub>mac</sub>239 and SIV<sub>smm</sub> (FYr1 and FWr1) were detected using plasma pooled from SIV-infected rhesus macaques and SIV-infected sooty mangabeys respectively. The Nef proteins of HIV-2 ROD10, ROD14, CBL-23 and 60415K were detected using plasma pooled from HIV-2-infected individuals. The Nef proteins of HIV-1 NL4-3 and NA7 were detected using polyclonal rabbit antisera. SIV p55 Gag, BST2 and β-actin were detected with the monoclonal antibodies 183-H12-5C, HM1.24 and C4. Following incubation with an appropriate HRP-conjugated secondary antibody, the blots were developed in chemiluminescent substrate and visualized using a Fujifilm Image Reader LAS 3000.</p

    SIV Nef downregulates rhesus tetherin, but not human tetherin.

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    <p>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<sub>2</sub>A and SIV Nef <i>crm<sup>−</sup></i> were expressed from the same mRNA transcript as GFP using pCGCG constructs in which expression of the <i>GFP</i> reporter gene was driven from an internal ribosomal entry site downstream of <i>nef</i>. Cells were also transfected with pCGCG without <i>nef</i> 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<sup>+</sup> cells for duplicate transfections.</p

    Dose-dependent reduction in the accumulation of SIV p27 in the cell culture supernatant relative to p55 Gag in cells at increasing expression levels of tetherin.

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    <p>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 <i>Δenv</i> and SIV Δ<i>nef</i> at increasing expression levels of human and rhesus tetherin. 293T cells were transfected with proviral DNA for wild-type SIV, SIV <i>Δenv</i> or SIV Δ<i>nef</i> 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, <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a>, 1997–2008) and compared as p27/p55 ratios. ‘ND’ indicates that the p27 band was below the limit of detection.</p

    Identification of the residues in rhesus tetherin required for recognition by SIV Nef.

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    <p>(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<sub>14</sub>DIWK<sub>18</sub> motif of rBST2 was also introduced into hBST2 (hBST2 G<sub>14</sub>DIWK<sub>18</sub>). 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 <i>Δnef</i> in cells expressing each of the rBST2 and hBST2 mutants shown in (A). Transfection and assay conditions were the same as previously described.</p

    Expression of the SIV envelope glycoprotein does not rescue virus release.

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    <p>The envelope glycoproteins of SIV<sub>mac</sub>239, HIV-2 ROD10 and HIV-2 ROD14 were tested for the ability to rescue virus release for SIV <i>ΔenvΔnef</i> in cells expressing human and rhesus tetherin. 293T cells were transfected with proviral DNA for SIV <i>ΔenvΔnef</i>, an expression construct for hBST2 or rBST2, and an expression construct for either SIV<sub>mac</sub>239 Env, HIV-2 ROD10 Env, HIV-2 ROD14 Env, SIV<sub>mac</sub>239 Nef or HIV-1 NL4-3 Vpu. The mean and standard deviation (error bars) are shown for total p27 release (A) and for percent maximal release (B). (C) Protein expression was confirmed for SIV Env, HIV-2 Env, SIV Nef, SIV p55 Gag and BST2 by western blot analysis of cell lysates. SIV Env was detected with a monoclonal antibody to SIV gp120 (KK42) and HIV-2 Env was detected with rabbit antisera to HIV-2 ST gp120. The SIV Nef protein was detected with the monoclonal antibody 17.2. SIV p55 Gag, BST2 and β-actin were detected with the monoclonal antibodies 183-H12-5C, HM1.24 and C4 respectively. Following incubation with an appropriate HRP-conjugated secondary antibody, the blots were developed in chemiluminescent substrate and visualized using a Fujifilm Image Reader LAS 3000.</p

    Interferon induces tetherin and inhibits the release of <i>nef</i>-deleted SIV from an infected rhesus macaque cell line.

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    <p>The rhesus macaque sMAGI cell line was cultured in the presence or absence of IFNα, and virus release was compared following infection with VSV G-pseudotyped SIV <i>Δenv</i> versus SIV <i>ΔenvΔnef</i>. (A) Treatment with IFNα upregulated the expression of tetherin on the cell surface. Cells were cultured in medium with or without 1000 U/ml IFNα, and the expression of tetherin was assessed by flow cytometry. (B) Deletion of the SIV <i>nef</i> gene significantly impaired virus release from infected cells in the presence of IFNα. Seventy-two hours after infection with VSV G-pseudotyped SIV <i>Δenv</i> and SIV <i>ΔenvΔnef</i> (50 ng/ml p27 eq. each), the amount of p27 released into the cell culture supernatant was determined by antigen capture ELISA.</p

    Species-specificity of HIV-1 Vpu and SIV Nef in counteracting restriction by human and rhesus tetherin.

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    <p>HIV-1 Vpu and SIV Nef were tested for the ability to rescue virus release in <i>trans</i> for SIV Δ<i>nef</i> and a <i>vpu</i>-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<i>nef</i> (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<sub>2</sub>A, SIV Nef <i>crm<sup>−</sup></i> or HIV-1 Nef. G<sub>2</sub>A and <i>crm<sup>−</sup></i> represent amino acid substitutions in the myristoylation site and the cholesterol recognition motif (L<sub>129</sub>R, Y<sub>133</sub>A and Y<sub>134</sub>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<sub>2</sub>A, Nef <i>crm-</i>, HIV-1 Nef and HIV-1 Vpu were verified by western blot analysis (I).</p
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