Contribution of flexible loop residues to Nef-mediated antagonism of tetherin and binding to AP-2.

<p>(A) Predicted amino acid sequence of the flexible loop region of the SIV_mac239 Nef protein. Substitutions at positions that impair anti-tetherin activity are underlined and in bold. The di-leucine and di-acidic motifs known to interact with AP-2 are indicated in brackets. (B) Tetherin downregulation by each of the flexible loop mutants was assessed by transfecting 293T cells that constitutively express HA-tagged rhesus tetherin with constructs expressing the indicated Nef mutants, and comparing the cell surface expression of tetherin to cells transfected with the empty vector (pCGCG). The percentage of tetherin expressed on the cell surface was calculated by dividing the MFI of tetherin (HA) staining on cells expressing Nef by the MFI of tetherin on cells transfected with the empty vector. (C and D) Histogram plots showing the surface expression of tetherin for cells expressing Nef mutants with substitutions in known AP-2 binding sites (C) and at sites flanking these residues (D). (E and F) Nef mutants with substitutions in the flexible loop were tested for binding to the α2 and μ2 subunits of AP-2 by co-immunoprecipitation. Parental 293T cells (E) or 293T cells that constitutively express HA-tagged rhesus tetherin (F) were transfected with constructs expressing the indicated Nef mutants, and cell lysates were immunoprecipitated with monoclonal antibodies to the α2 and μ2 subunits of AP-2. Western blots of immunoprecipitates and cell lysates were probed with antibodies to α2, μ2, Nef and tetherin. The ratios of the band intensities of Nef to α2 and Nef to μ2 in the immunoprecipitated samples are shown beneath each lane.</p

Distribution of Nef and tetherin in SIV-infected cells.

<p>293T cells expressing HA-tagged rhesus tetherin were infected with VSV-G pseudotyped SIV_mac239 Δ<i>env</i> and stained for tetherin (green), Nef (red) and the cell nucleus (blue). Tetherin expression in uninfected cells (A), and the localization of Nef and tetherin in SIV-infected cells (B). The white scale bar indicates 10 µm.</p

Identification of residues in SIV Nef necessary for tetherin antagonism.

<p>(A) Predicted amino acid sequence of SIV_mac239 Nef. The highlighted sequences correspond to the N-terminal domain (green), the globular core (blue), and the flexible loop region (orange). Substitutions in residues that impaired tetherin antagonism are underlined and in bold. (B, C and D) 293T cells were co-transfected with SIV Δ<i>nef</i> proviral DNA together with constructs expressing rhesus tetherin and either wild-type or mutant Nef proteins. The percentage of virus release was determined by measuring the accumulation of SIV p27 in the culture supernatant in the presence of tetherin relative to transfections with empty vector. Controls include virus release in the absence of Nef (pCGCG; white), wild-type Nef (black) and Nef with a glycine-to-alanine substitution in the myristoylation site (G₂A; red). Substitutions in the N-terminal domain are indicated by green bars (B), substitutions in the globular core domain are indicated by blue bars (C), and substitutions in the flexible loop region are indicated by orange bars (D). Error bars represent the standard deviation of duplicate transfections and the dotted line indicates 3 standard deviations over the activity of the G₂A mutant.</p

Subcellular distribution of tetherin in SIV-infected cells.

<p>293T cells expressing HA-tagged rhesus macaque tetherin were infected with VSV-G pseudotyped SIV_mac239 Δ<i>env</i> and stained for tetherin (green), Nef (blue) and either TGN46 (red) (A), CD63 (red) (B) or LAMP-1 (red) (C). The white scale bar indicates 10 µm. (D) The extent of co-localization between tetherin and each of the intracellular markers was estimated by calculation of the Pearson's correlation coefficients for images of twenty randomly selected SIV-infected cells.</p

CD4-downregulation, MHC class I-downregulation and infectivity enhancement mediated by SIV Nef mutants with impaired anti-tetherin activity.

<p>The surface expression of CD4 (A) and MHC class I (B) was compared on cells expressing each of the SIV Nef mutants. Jurkat cells were electroporated with a bicistronic vector (pCGCG) expressing either wild-type Nef or the indicated Nef mutants together with GFP. Cells were stained with a PerCP-conjugated monoclonal antibody to CD4 and an APC-conjugated monoclonal antibody to HLA class I molecules. Cells were gated on the GFP⁺ cell population and the percentage of CD4 and MHC I on the cell surface was determined relative to cells transfected with empty vector. Error bars indicate the standard deviation of duplicate transfections, and the black and red dotted lines correspond, respectively, to 3 and 5 standard deviations over the activity of wild-type Nef. (C) The Nef mutants with impaired anti-tetherin activity were also tested for infectivity enhancement. Viruses were generated by transient transfection of 293T cells with SIV_mac239 Δ<i>nef</i> proviral DNA and constructs expressing each of the Nef mutants, wild-type Nef or an empty vector. The infectivity of these viruses was then determined on GHOST X4/R5 cells, which express GFP upon SIV infection, 48 hours post-inoculation by flow cytometry. The relative infectivity of SIV Δ<i>nef trans</i>-complemented with the Nef mutants was calculated relative to SIV Δ<i>nef trans</i>-complemented with wild-type Nef. Virus infectivity in the absence of Nef is indicated by the white bars, and the activities mediated by wild-type Nef and the G₂A myristoylaton site mutant are indicated by black and red bars, respectively. Error bars indicate the standard deviation of duplicate infections, and the black and red dotted lines correspond, respectively, to 3 and 5 standard deviations over the infectivity of SIV Δ<i>nef</i> without <i>trans</i>-complementation. Substitutions in the N-terminal domain are indicated by green bars, substitutions in the globular core domain are indicated by blue bars, and substitutions in the flexible loop region are indicated by orange bars.</p

Summary of the properties of the SIV Nef mutants lacking anti-tetherin activity.

a<p>The percentage of BST-2 antagonism was calculated as the amount of SIV p27 released into the culture supernatant of 293T cells transfected with constructs expressing each of the Nef mutants in the presence of rhesus tetherin relative to the amounts of SIV p27 released in the absence of tetherin.</p>b<p>Binding to BST-2 was calculated as the relative band intensity of Nef to the band intensity of tetherin in immunoprecipitated samples.</p>c<p>Steady-state levels of Nef protein in cell lysates were estimated by calculating the relative band intensity of each of the Nef mutant proteins compared to wild-type Nef.</p>d<p>CD4 levels were calculated as the percentage of CD4 staining (MFI) on cells transfected with each of the indicated Nef-expression constructs relative to CD4 staining (MFI) on cells transfected with an empty vector.</p>e<p>MHC class I levels were calculated as the percentage of MHC I staining (MFI) on cells transfected with each of the indicated Nef-expression constructs relative to MHC I staining (MFI) on cells transfected an with empty vector.</p>f<p>GHOST X4/R5 cells were infected with SIV Δ<i>nef trans</i>-complemented with the indicated wild-type or mutant Nef proteins, and the relative infectivity was calculated as the frequency of infected cells obtained for each of the mutants relative to wild-type Nef at 48-hours post-inoculation.</p><p>Nef mutants that retain wild-type levels of CD4-downregulation, MHC class I-downregulation or infectivity enhancement are indicated in bold and italics. For CD4− and MHC class I-downregulation, one asterisk indicates activity within 5 standard deviations of wild-type Nef and two asterisks indicate activity within 3 standard deviations of wild-type Nef. For infectivity enhancement, two asterisks indicate infectivity 5 standard deviations or more over SIV Δ<i>nef trans</i>-complemented with empty vector.</p

Identification of residues in SIV Nef that contribute to interactions with tetherin.

<p>(A) Co-immunoprecipitation assays with the Nef mutants that impair anti-tetherin activity were performed to identify residues that diminish binding to tetherin. 293T cells were co-transfected with constructs expressing rhesus tetherin, and either wild-type or mutant Nef proteins. Cell lysates were immunoprecipitated using a monoclonal antibody to tetherin, and blots were probed with monoclonal antibodies to SIV Nef and tetherin. (B) Combinations of alanine substitutions in Nef that were shown to impair binding to rhesus tetherin in panel A were tested in additional co-immunoprecipitation assays as described above. (C) Estimated K_d.app values were determined for the binding of SIV_mac239 Nef_96–237 with the indicated deletions in the flexible loop region to the cytoplasmic domain of rhesus tetherin by SPR. Bands corresponding to the antibody heavy and light chain are indicated (HC and LC). Asterisks indicate the absence of detectable Nef protein.</p

Identification of residues in rhesus tetherin that contribute to interactions with Nef.

<p>Mutations in the cytoplasmic domain of rhesus and human tetherin were tested for their effects on binding to Nef by co-immunoprecipitation and SPR assays. (A) Co-immunoprecipitation of Nef in the presence of rhesus, human tetherin and the following tetherin mutants; a rhesus tetherin mutant lacking the first 10 residues of the protein (rΔ10), a rhesus tetherin mutant containing five alanine substitutions at positions 14–18 (rA₁₄-A₁₈) and a human tetherin mutant containing residues D₁₄DIWK₁₈ from rhesus tetherin (hDDIWK). The ratios of the band intensities of Nef versus tetherin in the immunoprecipitates are shown beneath each lane. (B) SPR assays were performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003487#ppat-1003487-g001" target="_blank">Figure 1</a> to assess binding for Nef_96–237 and peptides corresponding to the tetherin mutants tested in panel A. (C) Virus release assays were performed to test the susceptibility of each of these tetherin mutants to Nef antagonism. 293T cells were co-transfected with SIV_mac239 Δ<i>nef</i> proviral DNA and constructs coding for rhesus, human tetherin, each tetherin mutant or an empty vector. Constructs coding for Nef were provided <i>in trans</i>. Virus release was measured by SIV p27 antigen-capture ELISA and expressed as the percentage of maximal release in the absence of tetherin.</p

Tetherin antagonism by Nef depends on clathrin and dynamin 2.

<p>(A) Dominant-negative mutants of AP180 and Dyn2 were tested for disruption of Nef-mediated tetherin downregulation. 293T cells expressing HA-tagged rhesus tetherin were transfected with constructs expressing Nef, or empty vector, and constructs expressing AP180C, Dyn1K44A, Dyn2K44A and Dyn2. Surface expression of tetherin was measured by HA staining, and the percentage of tetherin expressed on the cell surface was determined by dividing the MFI of tetherin on cells expressing the dominant-negative mutants by the MFI of tetherin on cells transfected with empty vectors. (B) The dominant-negative mutants of AP180 and Dyn2 were also tested for their effects on Nef-mediated virus release. 293T cells were transfected with SIV_mac239 or SIV_mac239 Δ<i>nef</i> proviral DNA, and constructs expressing rhesus tetherin, and either AP180C, Dyn1K44A, Dyn2K44A, Dyn2 or empty vector. Virus release was measured by SIV p27 antigen-capture ELISA and expressed as the percentage of maximal release in the absence of tetherin. (C) Protein expression for Nef, tetherin, AP180C, Dyn1K44A, Dyn2K44A and Dyn2 was verified by western blot analyses using endogenous β-actin as a loading control. (D) Replication of wild-type SIV (WT SIV) versus SIV Δ<i>nef</i> in the presence and absence of Dynasore. 221 T cells were infected with 20 ng p27 of wild-type SIV or SIV Δ<i>nef</i>. Twenty-four hours post-infection, cells were treated with IFNα (100 U). Eight hours later, Dynasore (20 µM) was added to one of the cultures. Supernatants were collected at the indicated time points and virus replication was determined by SIV p27 antigen-capture ELISA.</p

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

<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 G₁₄DIWK₁₈ motif of rBST2 was also introduced into hBST2 (hBST2 G₁₄DIWK₁₈). 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