The effects of N-terminal insertion into VSV-G of an scFv peptide

Table 1 Infection assay on human cells.

Virus	Exp 1	Exp 2	Exp 3	Exp 4	Exp 5	Exp 6	Exp 7
αMHC/VSV-G	7200	3020	3620	2880	1400	1720	1800
αHEL/VSV-G	1120	620	760	380	260	360	420

Supernatant from HIV-1-producing 293T cells were passed through a 0.45 μm filter (Sarstedt). αMHC/VSV-G and αHEL/VSV-G HIV-1 samples were concentrated 100 times by centrifugation (25 kRPM at 4°C for 2 hours in a BeckmanCoulter ultracentrifuge) and were resuspended in 1% bovine serum albumine. VSV-G pseudotyped HIV-1-derived viral particles were directly used without prior concentration to infect target cells. When required, the virus was stored at -80°C. 50% confluent target cells, either human 293T (depicted) and HeLa cells, mouse Mus Dunni cells or monkey Cos-7 cells (not shown), were cultured with dilutions of virus for 16 hours in the presence of 5 μg/ml polybrene. 48 hours later, green fluorescent colonies were counted. Titres (colony forming units (cfu)/ml) on infected 293T cells of αMHC/VSV-G and αHEL/VSV-G particles from 7 independent experiments are shown. VSV-G pseudotyped HIV-1 was used as control for successful virus production and infection, and generally gave titers of 10⁷-10⁶ cfu/ml (data not shown).

ISSN: 1743-422X