0.45 μm pore membrane

Linear polyetylenimine (PEI, Polysciences) was used to co-transfect HEK293T cells with the barcoded transfer plasmid pHCC1, Δ8.91 packaging plasmid and a vesicular stomatitis virus G (VSV-G) protein encoding plasmid to produce VSV-G-pseudotyped vectors. The barcoded plasmid pHCC1 was produced as described before (43 (link)). Six hours post transfection, cells were washed twice with phosphate buffered saline (PBS) to remove the excess of plasmid. Supernatant was collected 72 h post-transfection and filtered through a 0.45 μm pore membrane (Merck, Overijse, Belgium). The vector was concentrated using a Vivaspin with a 15–50 kDa cut-off column (Merck) and washed three times with PBS. Next, the vector was treated with 100 U/ml DNase (Roche Diagnostics, Vilvoorde, Belgium) for 1 h at 37°C and stored at −80°C.

The CC81-GREMG cell line is derived from the mouse CC81 sarcoma virus-transformed feline cell line and expresses enhanced green fluorescent protein (EGFP) in a BLV Tax-inducible manner [26 (link)]. CC81-GREMG-CAT1 was established by stably transducing CC81-GREMG cells with the bovine CAT1 gene [27 (link)]. FLK-BLV is a sheep cell line with persistent BLV infection. Both cell lines were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), as described previously [26 (link),27 (link)]. To prepare infectious BLV particles, FLK-BLV cells (1 × 10⁶ cells) were cultured for 4 days, and the culture supernatant was collected, centrifuged at 440× g for 15 min to remove cell debris, and filtered through a 0.45 μm pore membrane (Merck Millipore, Darmstadt, Germany). The virus-containing supernatant was stored at −80 °C until use. For the binding analysis, virus particles were concentrated by centrifuging the culture supernatant at 42,200× g for 2 h at 4 °C, and the pellet was suspended in a 1/10 volume of fresh medium. The liquid sample containing the concentrated virus was aliquoted and stored at −80 °C until use.