SUPPLEMENTARY FIGURE LEGENDS
Supplementary Fig. 1. Alphavirus vectors used in this study. SFV-LacZ, SFV-helper-
CS219A and SIN-GFP have been previously described [supplementary refs. 1-3]. Black arrows indicate the position of the subgenomic promoter from which Rep starts synthesis of sgRNAs. The solid orange circle to the left of each RNA represents CAP. nsp, Rep non- structural protein subunits; b1 and b7, SFV translation enhancers coding for the first 34 or 44 amino acids of the capsid, respectively; 2A, foot and mouth disease virus 2A autoprotease
[supplementary refs. 4,3] (a sequence of 17 amino acids which cleaves in cis between residues
16 and 17, as indicated by the gray arrow), Capsid*, SFV capsid containing mutation S219A, which abolishes its proteolytic activity [supplementary ref. 3].
Supplementary Fig. 2. Expression of envelope proteins in transfected cells. BHK cells were electroporated with RNAs from the indicated vectors and after 12 h (a) or 6 h (b) cell lysates were analyzed by Western blot with an antiserum specific for SFV envelope proteins
(upper gel in a and b), capsid (intermediate gel in a), -actin (lower gel in a) or the nsP1 subunit of Rep (lower gel in b). The small protein 6K was not detected with the anti-spikes antiserum used in this assay, but absence of 6K expression in cells electroporated with SFV- enh-spikeΔ6K RNA was confirmed by metabolic labeling experiments (data not shown). e- spike, SFV-enh-spike; spike, SFV-spike; LacZ, SFV-LacZ; wt, wtSFV; Δ6K, SFV-enh- spikeΔ6K; mock, mock- electroporated cells.
Supplementary Fig. 3. Propagation of SFV without capsid is not mediated by syncytia formation. BHK cell monolayers were infected with the indicated vectors and at 16 h.p.i. were incubated with PBS at pH 7 or 5 as described in legend for Fig. 3b. Three hours later,
1 cells were fixed with 4% paraformaldehyde and stained with 5 g/ml wheat germ agglutinin
(WGA) conjugated with Alexa Fluor® 488 (Life Technologies) to stain cellular plasma membranes. Cells were then permeabilized with 0.1% Triton X-100 and analysed by immunofluorescence with a polyclonal antiserum specific for the nsP2 subunit of SFV Rep using as secondary antibody anti-rabbit IgG serum conjugated with C3 (Sigma). Images were taken with the appropriate filters and merged using Image J 1.48v (NIH, MD). Magnification of images is 1000x; scale bars: 10 m. Images of a representative experiment performed in triplicate are shown.
Supplementary Fig. 4. Infectious material can transfer viral RNA to newly infected cells.
Infectious material released by SFV-enh-spike-transfected HuH-7 cells was used to infect
BHK cells monolayers at MOI 0.01 and viral RNA present in cells was analyzed at the indicated times by quantitative RT-PCR, using oligonucleotides specific for SFV Rep as described in Materials and Methods. wtSFV infected cells were analyzed at 4 h.p.i., because at 6 h.p.i. there was already some degree of propagation (see Figure 1b), something that was not observed at 4 h.p.i. (data not shown). Results correspond to one out of two experiments, each performed in triplicate, that gave very similar results. The numbers above the 24 h bars indicate the fold increase in viral RNA levels between the two indicated time points. Error bars represent the mean + SD. **p<0.01; ***p<0.001. ns, not significant; nd, not detected.
Supplementary Fig. 5. Analysis of viral and cellular proteins in iMVs. HuH-7 cells were electroporated with the indicated RNAs, and iMVs were purified as described (iMVs).
Lysates from the same cells were obtained at 24 h post-electroporation (Lysates). Samples were analyzed by Western blot with antisera specific for the indicated cellular (a and b) and viral (a and c) proteins. Gels were loaded with approximately 5x104 IU of iMVs (e-spike) or
2 an equivalent volume of material purified from SFV-LacZ (LacZ) or mock-electroporated cells (mock). The amount of lysate from SFV-enh-spike electroporated cells was adjusted to produce a signal similar to that of 5x104 IU of iMVs. Exo, exosomes purified from the supernatant of untransfected HuH-7 cells as described in Materials and Methods. For this analysis at least three independent stocks of iMVs were analyzed obtaining very similar results. Gels show the analysis of a representive stock.
Supplementary Fig. 6. Characterization of biotinylated iMVs. HuH-7 cells were electroporated with SFV-enh-spike RNA, and approximately 5x106 cells from the same electroporation mix were plated in parallel dishes. After o/n incubation cells were labeled with [35S] methionine-cysteine at 100 mCi/ml (PerkinElmer, Waltham, MA) for 30 min., and chased for 50 min. Next, electroporated cells were either derivatized with biotin (Life
Technologies) or mock derivatized and then incubated for 18 h at 37°C as described
[supplementary ref. 5]. Cell supernatants and lysates were then collected for analysis.
Equivalent aliquots of the supernatant were retrieved with streptavidin-coated magnetic beads
(Dynabeads® MyOne™ Streptavidin T1, Life Technologies) and directly analyzed by SDS-
PAGE (the amount loaded per lane corresponds to material purified from 5x106 cells). Cell lysates were analyzed directly by SDS-PAGE, and were used to control that transfection and labelling had been similar in biotin- and mock-derivatized cells (the amount loaded per lane corresponds to 105 cells). Notice that the Supernatants´ gel was exposed 4-times longer than the Lysates´ one. A representative experiment is shown.
Supplementary Fig. 7. Analysis of budding of iMVs by TEM. Representative images of thin-section TEM of BHK cells infected with the indicated vectors at MOI 10 and analyzed at
16 h. p. i. Samples for thin-section TEM were prepared as described [supplementary ref. 6]
3 Black arrows within insets indicate putatitve iMVs (upper image) and wtSFV VPs (lower image) being released from infected cells. Magnification of large images: 2520x; insets:
20,660x.
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