Fig 4.

B19V and the VP1u region share the same internalization pathway. (A) Unlabeled recombinant ΔC128 VP1u (50 ng) alone or in the presence (+) of a 12-fold excess of N-VP1u Ab was incubated with UT7/Epo cells for 1 h at 4°C. Cells were washed, and cell-bound VP1u was detected by IF (anti-FLAG Ab) and WB (anti-PLA₂ Ab). (B) ΔC128 VP1u was internalized alone or in the presence of N-VP1u Ab for 30 min at 37°C. Cells were briefly trypsinized, and internal VP1u was detected by IF and WB. (C) B19V internalization into UT7/Epo cells for 30 min was carried out in the presence (+N-VP1u Ab) or absence (w/o Ab) of 0.4 μg of N-VP1u Ab. Internal capsids were stained with anticapsid Ab and detected by IF. In parallel, DNA of internalized virions was extracted and quantified by qPCR. (D) Prior to B19V internalization, UT7/Epo cells were incubated for 1 h at 4°C with recombinant VP1u proteins (150 ng of WT VP1u or ΔN29 VP1u; 75 ng of ΔC128 VP1u) corresponding to a 50-fold excess to the applied B19V. After 30 min at 37°C, internalized B19V was detected by IF and qPCR. Values of internalized virions were normalized to the value of internalization without Ab or recombinant VP1u. The sample at 4°C (no internalization) demonstrates the efficient removal of particles by trypsinization when internalization does not occur.