TYTLE/5A mutation of F is detrimental for SeV particle rescue.

<p><b>A</b>. Coomassie blue stained PAGE analysis of virus purified from egg allantoic fluids. Lanes P2, P3, P4 (HA-F_5A/Fgfpt), 2^nd to 4^th egg to egg serial passages of rSeV-HA-F_5A/Fgfpt rescue. Lane P2 (HA-F/Fgfpt), 2^nd passage of rSeV-HA-F/Fgfpt rescue. <b>B</b>. Western blot analysis of MDCK cellular extracts obtained 24 hours after mock infection (Mock), infections with rSeV-HA-F/Fgfpt (HA-F/Fgfpt) or rSeV-HA-F_5A/Fgfpt (HA-F_5A/Fgfpt) in conditions of suppression or not of the endogenous wt-F proteins (+/− α-gfpt siRNA). α-Fct : anti Fct antibody. α-HA: anti HA tag antibody. <b>C</b>. The cDNA fragment containing the HA-F_5A gene was purified after MluI digestion of the rSeV-HA-F_5A/Fgfpt full length cDNA clone and fully sequenced. <b>D</b>. The viral genomic RNA present in rSeV-HA-F_5A/Fgfpt infected cells was reverse transcribed using a primer of positive polarity in the M gene. The RT product was amplified using a primer of negative polarity in HA tag sequence. The RT-PCR product was then purified and fully sequenced. Arrows in <b>C</b>. and <b>D</b>. indicate the G to T substitution position leading to introduction of a STOP at position 37 in the F ORF (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078074#pone.0078074.s001" target="_blank">figure S1</a>).