(A) Alignment of the cytoplasmic tails of hu-tetherin and rh-tetherin-1 N-terminal cytoplasmic tails with differences in rh-tetherin-1 highlighted. Also shown are the three mutant hu-tetherin constructs that were used.
(B) Western blot analysis of the expression of the mutant hu-tetherin proteins and inhibition of SIV_MAC239(del Nef) particle release. Cells were transfected with a fixed amount (500ng) of proviral plasmid and 0, 12.5ng, 25ng, 50ng, 100ng or 200ng of each tetherin expression plasmid. Blots of cell lysates were simultaneously probed with rabbit anti-HA and (upper panels) and anti-CA monoclonal antibodies (middle panels). Lysates of pelleted virions were probed with anti-CA antibodies only. Signals were detected using fluorescent detection reagents (LICOR).
(C) Same as (B), except that SIV_MAC239(WT) was used in place of SIV_MAC239(del Nef).
(D) Quantitative analysis of virion associated p27CA, determined using the western blots shown in (B) for SIV_MAC239(del Nef) and (C) for SIV_MAC239(WT).
Results are representative of at least three independent experiments.

(A) Rhesus macaque 221 cells and human 293T cells were infected with VSV-G pseudotyped SIV_MAC239(WT) or SIV_MAC239(del Nef), as indicated, at an MOI of 0.2 for 24h, then washed and incubated with 0, 10, 100 or 1000U/ml of IFNα for 24h. Cell and virion lysates were probed with anti-CA antibodies and signals revealed using fluorescence detection (LICOR, see Figure S6A, B for quantitation).
(B) Rhesus macaque 221 cells were infected with VSV-G pseudotyped SIV_MAC239(WT) (i and iii) or SIV_MAC239(del Nef) (ii and iv), as indicated, at an MOI of 2 for 24h, then washed and incubated in the absence (i and ii) or presence (iii and iv) of 1000U/ml of IFNα for 24h. Cells were then fixed and examined using transmission electron microscopy. Scale bar = 500nm. (iv) inset, shows an expanded view of virions apparently tethered to each other.
(C) African green monkey COS-7 cells were infected with VSV-G pseudotyped HIV-1 encoding no Nef, HIV-1 Nef or SIV_AGMSab Nef, as indicated, at an MOI of 1 for 24h, then washed and incubated with 0, 10, 100 or 1000U/ml of IFNα for 24h. Cell and virion lysates were probed with anti-CA antibodies and signals revealed using fluorescence detection (LICOR, see Figure S6C for quantitation of virion release).
Results are representative of at least three independent experiments.

(A–E) Western blot analysis of HIV-1 particle release. A fixed amount (200ng) of each proviral plasmid was co-transfected with a fixed amount (12.5ng) of rhesus macaque (rh-1), pig-tailed macaque (pgt), human (hu) or African green monkey (agm) tetherin-HA expression plasmids, along with varying amounts (0ng, 25ng, 50ng, 100ng) of HIV-1 Nef (A), SIV_MAC Nef (B), SIV_AGMSab Nef (C), SIV_AGMTan Nef (D), or SIV_BLU Nef (E) expression plasmids. Cell and virion lysates were probed with an anti-capsid monoclonal antibody. Samples derived using each Nef protein were run on a single gel, vertical lines are for visual guidance only. Left panels are film images following detection using chemiluminescence reagents, right panels show the results of p24CA quantitation in virion samples measured using quantitative fluorescence-based western blotting (LICOR). Results are from a single experiment and are representative of at least three separate experiments.

(A) Cells were transfected with 400ng of an HIV-1 proviral plasmid lacking Nef and Vpu, along with 25ng of the rh-tetherin expression plasmid and 100ng of each WT or mutant SIV_MAC239 Nef expression plasmid. Blots of cell lysates were simultaneously probed with rabbit anti-HA and (upper panels) and anti-CA monoclonal antibodies (middle panels). Lysates of pelleted virions were probed with anti-CA antibodies only (lower panels). Signals were detected using quantitative fluorescent western blotting (LICOR) and numbers below each lane represent measurements of virion associated p24.
(B) Cells were transfected as in (A) and the yield of infectious virions in the culture supernatant was measured using TZMbl indicator cells. Measurements are given in relative light units (RLU) following detection of β-galactosidase expression using a chemiluminescent assay. Results are the mean± SD of tripicate determinations from a single experiment and are representative of two independent experiments.
(C) Cells were transfected with 400ng of an HIV-1 proviral plasmid lacking Nef and Vpu, along with 25ng of the rh-tetherin expression plasmid and 50ng of each HIV-2, SIV_SMM or SIV_MAC239 Nef expression plasmid. Blots were probed and analysed as in (A) and numbers below each lane represent measurements of virion associated p24.
(D) Cells were transfected as in (C) and the yield of infectious virions in the culture supernatant was measured as in (B). Results are the mean± SD of tripicate determinations from a single experiment and are representative of two independent experiments.

(A–H) Western blot analysis of HIV-1 particle release. A fixed amount (200ng) of each proviral plasmid was co-transfected with empty vector (none) or with increasing amounts (3.12ng, 6.25ng, or 12.5ng, left to right) of African green monkey (agm), human (hu), pig-tailed macaque (pgt), or rhesus macaque (rh-1) tetherin-HA expression plasmids. Cell and virion lysates were probed with an anti-capsid monoclonal antibody. Proviral plasmids carrying neither Nef nor Vpu (A) or HIV-1 Vpu (B) were used as controls. Thereafter, proviral plasmids lacking Vpu, but encoding HIV-1 Nef (C), SIV_MAC Nef (D), SIV_AGMSab Nef (E), SIV_BLU Nef (F), SIV_RCM Nef (G), or SIV_GSN Nef (H) were used. Samples derived from each viral construct were run on a single gel, vertical lines are for visual guidance only. Left panels are film images following detection using chemiluminescence reagents, right panels show the results of p24CA quantitation in virion samples measured using quantitative fluorescence-based western blotting (LICOR). Results are from a single experiment and are representative of at least three separate experiments.

(A) Schematic representation of the intact and chimeric tetherin proteins generated by exchange of sequences encoding the hu-tetherin and rh-tetherin cytoplasmic domains, generating huCT-rh-tetherin and rhCT-hu tetherin.
(B) A fixed amount of (500ng) an HIV-1 proviral plasmid lacking both Vpu and Nef (upper panels) or lacking Vpu and encoding SIV_MAC Nef (lower panels) was transfected either alone (− lanes) or along with 50ng of each intact or chimeric tetherin-HA expression plasmid depicted in (A) (+ lanes). The panels show western blots of cell and virion lysates probed with anti-capsid or anti-HA monoclonal antibodies, and detection with chemiluminescence based reagents. Numbers below each lane represent measurements of virion associated p24 measured using quantitative fluorescent western blotting (LICOR).
(C) An HIV-1 proviral plasmid carrying SIV_MAC Nef (500ng) was cotransfected with increasing amounts (0ng, 50ng, 100ng, 200ng) of each tetherin expression plasmid depicted in (A). Infectious virion yield was measured as in Figure 3. Results are the mean± SD of tripicate determinations from a single experiment and are representative of three independent experiments
(D) Western blot analysis of SIV_MAC239(WT) and SIV_MAC239 (delNef) particle release in the presence of the intact and chimeric tetherin-HA proteins. Cells were transfected with a fixed amount (500ng) of each SIV_MAC239 proviral plasmid and varying amounts (0, 50ng, 100ng) of each tetherin expression plasmid. The panels show western blots of cell and virion lysates probed with anti-capsid or anti-HA monoclonal antibodies, and detection with chemiluminescence based reagents. Numbers below each lane represent measurements of virion associated p27 measured using quantitative fluorescent western blotting (LICOR).
(E) An SIV_MAC239(WT) proviral plasmid was cotransfected with increasing amounts (0ng, 13ng, 25ng, 50ng, 100ng, 200ng) of each intact or chimeric tetherin expression plasmid depicted in (A). Infectious virion yield in the culture supernatant was measured as in Figure 3. Results are the mean± SD of tripicate determinations from a single experiment and are representative of three independent experiments

(A) Western blot analysis of wildtype (WT) and envelope deleted (del Env) SIV_MAC239 particle release in the presence of tetherin-HA proteins from rhesus (rh) and pig-tailed macaques (pgt). Cells were transfected with 400ng of proviral plasmid and 40ng of each tetherin expression plasmid. Cell and virion lysates were probed with an anti-capsid monoclonal and/or rabbit anti-HA antibody, and signals detected using fluorescent secondary antibodies. The numbers below each lane indicate quantitation (LICOR) of the p27CA present in the virion pellet.
(B,C) Western blot analysis of wildtype (WT) and Nef-deleted (del Nef) SIV_MAC239 particle release in the presence of the hu-tetherin (B) or rh-tetherin-1 (C) proteins. Cells were transfected with a fixed amount (400ng) of proviral plasmid and varying amounts (50, 25, 12.5, 6.25, 3.12 or 0ng) of each tetherin expression plasmid. Cell and virions lysates were probed with an anti-capsid and anti-HA epitope monoclonal antibodies, and signals detected using chemiluminescence reagents. The results shown are representative of at least three separate experiments.
(D) Quantitation of virion release inhibition by tetherin. P27CA in virion samples from panel (B) and (C) was measured using quantitative fluorescence-based western blotting (LICOR).
(E) Representative scanning EM images of cells transfected with pSIVGag-IRESGFP and either no tetherin, rh-tetherin-1 or hu-tetherin. Cells were selected for imaging based on similar levels of GFP fluorescence. Scale bar indicates 2µm.
(F) Representative fluorescence micrographs of unmanipulated or rh-tetherin-1 expressing 293T cells transfected with plasmids expressing SIV_MAC239 Gag-GFP fusion protein, in the presence or absence of SIV_MAC239 Nef.
(G) Quantitation of the proportion of cells displaying both plasma membrane and internal (PM+Int) or plasma membrane only (PM) intense accumulations of SIV_MAC239 Gag-GFP, under the same experimental conditions as in (F) (see (F) for examples). Cells were transfected with 0µg (−), 0.8µg(+) or 1.6µg(++) of pCR3.1/Nef. Values represent the mean ± SD of triplicate determinations and are representative of two independent experiments. At least 100 individual cells were evaluated under each condition.