In fact, we observed reduced fiber trimerization for chimeric Bekanamycin viruses with long shaft, indicating that trimerization is hampered when fusing the HAdV-41 short fiber knob with the HAdV-5 shaft. As only trimeric fibers can be incorporated into viral particles, is it somewhat surprising that Ad5TS*/41sSK and Ad5TS/41sSK fibers with YSA peptide inserted into the IJ loop show normal fiber content in purified viruses. This result shows that even low-level trimerization can be sufficient to ensure fiber incorporation. However, incorporation of fibers into viral particles was reduced or lost after YSA peptide insertion into the EG or HI loops, respectively. Trimerization and incorporation deficiency of the Ad5TS/41sSK-HI-YSA fiber is in contrast to results for the HAdV-5 fiber, which is susceptible to YSA peptide insertion into the HI loop. This difference can be explained by the different sequence, length and intramolecular environment of the HI loops in the HAdV-41 short fiber versus the HAdV-5 fiber. However, note that short shafted HAdV-41 fibers accepted YSA insertions into the HI loop without loss of trimerization and incorporation, because of the above mentioned critical role of the shaft domain for fiber trimerization and incorporation into virus particles. Ad5TS/41sK viruses with YSA peptide inserted into the EG loop and especially into the IJ loop resulted in significant transduction of EphA2-negative cells. This property might be independent of the inserted ligand, as Nakamura et al. previously reported increased Ad transduction in vitro and in vivo for the HAdV-40 short fiber knob when fused to a long fiber shaft. Possible explanations are direct cell Nomifensine Maleate binding via the shaft domain or shaft length-dependent modification of cell binding properties of the knob. The latter was shown for CAR-binding knobs, which mediate strongly reduced adenoviral transduction when fused to a short shaft. Finally, YSA-mediated viral transduction was lost irrespective of the peptide insertion site, when a HAdV-5 fiber shaft containing a mutation of the putative HSPG binding motif was used, which is in accord with previous studies and is hypothesized to result from reduced shaft flexibility and/or defective post-entry virus trafficking.