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. Author manuscript; available in PMC: 2016 Dec 20.
Published in final edited form as: Nat Microbiol. 2016 Jun 20;1(8):16080. doi: 10.1038/nmicrobiol.2016.80

Figure 5.

Figure 5

Anti-IAV VHHs block nuclear import of vRNPs and mRNA transcription. (a, b) A549 cells or clones expressing the indicated VHHs were treated with 1 μg/mL Dox for 24 h and infected with IAV WSN (MOI 230) in the presence of 1 mM cycloheximide for 4 h. Controls were treated with 50 nM bafilomycin A1 (BafA). Cells were stained for NP, HA, DNA, and actin; Z-stacks were recorded by confocal microscopy and Z projections of representative examples are displayed in (a). Scale bars represent 20 μm. NP staining in the nucleus and cytoplasm was quantified with CellProfiler and ratios of nuclear/cytoplasmic signal intensities were quantified and normalized to untreated cells (nuclear import = 1.0) and BafA-treated cells (nuclear import = 0). Values from three independent experiments ± s.e.m. are shown. (c, d) 293T cells were transfected with expression vectors for IAV WSN PA, PB1, PB2, NP, pPolI-EGFP, and the indicated HA-tagged VHHs. 24 h post transfection, cells were stained for HA and analyzed by flow cytometry. The fraction of VHH-HA-positive cells that expressed high levels of EGFP was quantified. Exemplary histograms are shown in (c), and average data from three independent experiments ± s.e.m. are displayed in (d).