HAdV-C5 viruses pseudotyped with the HAdV-D26 or HAdV-D35 fiber knob proteins were generated by the recombineering method, as published by Stanton et al. (28). In brief, a marker cassette was generated using the SacB cassette template (28), with the SacB primer pair (table S1) with homology to the HAdV-C5 fiber knob DNA sequence before the Threonine, Leucine, Tryptophan (TLW) hinge sequence (forward primer) and after the stop codon (reverse primer). This template was integrated into a bacterial artificial chromosome (BAC) containing the genome of a GFP-expressing HAdV-C5, which had been rendered replication incompetent by deletion of the E1A gene, now containing a marker cassette instead of the HAdV-C5 fiber knob domain.

The DNA sequence of the HAdV-D26K and HAdV-B35K fiber knobs was amplified using the 26K and 35K primer pairs, respectively (table S1), containing similar homology to the SacB cassette. A second round of recombineering was used to generate the final HAdV-C5 pseudotyped genomes by integrating the HAdV-D26K and HAdV-B35K polymerase chain reaction (PCR) transcripts. After recombineering, the new BACs were sequenced to confirm the correct fiber knob DNA sequence.

The BAC DNA for the new vector genomes was transfected into 293 cell line stably expressing the tetracycline repressor protein (TREx-293) cells (1.5 × 106), cultured in a T25 cell bind flask (Corning) in 5 ml of Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum, using the effectene system (QIAGEN). Cells were kept in culture until a cytopathic effect (CPE) was observed, at which point they were harvested by scraping and centrifugation at 1200g for 3 min. Cells were resuspended in 1 ml of media and frozen at −80°C to create a crude stock of virus. TREx-293 cells were cultured at 70% confluency in 5× T150 cell bind flasks (Corning) containing 20 ml of complete DMEM and then inoculated with 10 μl of crude virus stock. Cells were maintained in culture until CPE was observed and harvested by scraping and centrifugation at 1200g.

Virus was then purified from this cell pellet using the Cesium Chloride (CsCl) gradient method (52). Virus titer was determined in viral particles per milliliter (VP/ml) using the Pierce BCA Protein Assay Kit, assuming 4 × 109 VP/μg of protein (52). By using this method, we were able to generate HAdV-C5 viruses pseudotyped with the fiber knob domains of HAdV-D26 or HAdV-B35. These viruses retained the HAdV-C5 fiber shaft domain and were replication incompetent.

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