Phenotypic lentivirus screens to identify functional single domain antibodies

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1 ARTICLE NUMBER: DOI: /NMICROBIOL Phenotypic lentivirus screens to identify functional single domain antibodies Florian I. Schmidt, Leo Hanke, Benjamin Morin, Rebeccah Brewer, Vesna Brusic, Sean P.J. Whelan, and Hidde L. Ploegh Supplementary Figure 1 Lentivirus transduction efficiency in screens. A549 cells were transduced with the lentivirus library at a multiplicity of infection (MOI) of 0.25 in the presence of 10 µg/ml polybrene. expression was induced 8 h post transduction by the addition of doxycycline (Dox) to a final concentration of 1 µg/ml. 48 h post transduction, cells were harvested, stained with anti-neomycin phosphotransferase II (NPII), and analyzed by flow cytometry. The gated fraction of cells was corrected by the value obtained from mock-transduced A549 cells to obtain the final fraction of transduced cells. NATURE MICROBIOLOGY 1

2 DOI: /NMICROBIOL Supplementary Figure 2 Expression of antiviral s. A549 cells or clones inducibly expressing the indicated s were treated without or with 1 μg/ml Dox for 24 h, infected with IAV WSN for 6 h, stained for -HA expression, and analyzed by flow cytometry. All data is from three independent experiments ± s.e.m (same data set as figures 3c and 3d). Supplementary Figure 3 Validation of antiviral s. A549 cells were transduced with lentivirus encoding the indicated s in the presence of 10 µg/ml polybrene. expression was induced 8 h post transduction by the addition of Dox to a final with 1 μg/ml Dox. 48 h post transduction, cells were infected with IAV WSN (a) or VSV Indiana EGFP (b) for 6 or 4 h, respectively. IAV-infected cells were stained for NP expression. Cells were analyzed by flow cytometry and the absolute fraction of infected cells was determined. Data from one experiment representative of two independent repetitions is shown. 2 NATURE MICROBIOLOGY

3 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 4 Competition assay to determine epitope bound by NP2. Purified IAV NP was pre-incubated with the His 6 -tagged s indicated at the top and subsequently subjected to immunoprecipitation with biotinylated NP2. Precipitation of NP and the respective was analyzed by SDS-PAGE and colloidal Coomassie staining. Competition due to overlapping binding epitopes of the s is indicated with red squares, successful co-purification with green squares. 69 contains CDR regions nearly identical to 22 and is for this reason no longer shown in the main figures (as the respective cluster is represented by 22). Data representative of three independent experiments is displayed. NATURE MICROBIOLOGY 3

4 DOI: /NMICROBIOL Supplementary Figure 5 Nuclear import of transiently expressed NP in presence of NP-specific s. 293T cells were transfected with expression vectors for IAV WSN PA, PB1, PB2, NP, ppoli-egfp, and the indicated HA-tagged s. 24 h post transfection, cells were fixed, stained for HA and NP, and imaged by spinning disk confocal microscopy. Z projections of representative cells from three experiments are displayed; scale bars represent 10 μm. 4 NATURE MICROBIOLOGY

5 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Supplementary Figure 6 IAV minigenome assay. 293T cells were transfected with expression vectors for IAV WSN PA, PB1, PB2, NP, and ppoli-egfp, or a combination of plasmids lacking the indicated vector. 24 h post transfection, cells were harvested and analyzed by flow cytometry. A histograms representative of two independent experiments is shown. Supplementary Figure 7 Complete gels and autoradiograms. Complete Coomassie-stained gels from experiments shown in figures 4b, 4d, and supplementary figure 4, as well as complete autoradiograms from experiments in figures 6a and 6c are shown. NATURE MICROBIOLOGY 5

6 DOI: /NMICROBIOL Full gel pictures corresponding to figure 4b [kd] NP1 HA IP with NP1-biotin NP [kd] NP1 HA IP with 22-biotin NP [kd] NP1 HA IP with 1-biotin NP 6 NATURE MICROBIOLOGY

7 DOI: /NMICROBIOL SUPPLEMENTARY INFORMATION Full gel pictures corresponding to supplementary figure 4 [kd] NP1 NP NP IP with NP2-biotin IAV NP competition assays Full gel pictures corresponding to figure 4d IP with -biotin N NATURE MICROBIOLOGY 7

8 Full autoradiogram corresponding to figure 6a DOI: /NMICROBIOL L - G - N - P/M - Full autoradiogram corresponding to figure 6c L - G - N - P/M - no NP1 8 NATURE MICROBIOLOGY