The amount of M, F and G plasmids used at transfection has a bearing on the level of particle formation based on minigenome-encoded reporter gene levels in VLP-infected cells.

<p>Cells were transfected with increasing concentrations of M, <u>or</u> F <u>or</u> G expression plasmids (indicated by a triangle) while keeping the concentration of the other two plasmids fixed. They were co-transfected with the previously optimized minigenome and N, P and L constructs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018437#pone.0018437-Harcourt2" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018437#pone.0018437-Walpita1" target="_blank">[57]</a>. Forty eight hours post transfection, the cell SUPs were clarified by centrifugation, and same volume of SUP from each sample was used to infect new cell monolayers (VLP infected) which were transfected 24 hours previously with the core plasmids N, P and L to support replication of the VLP-incorporated minigenome RNA; the VLP-infected cells were harvested 48 hours later for reporter gene analysis. <b>Panel A</b> shows the plasmids transfected in each reaction. <b>Panel B</b>: shows minigenome-encoded CAT activity in VLP infected cell monolayers. Lane 1 is a negative control. Absence of CAT activity in duplicate lanes 2 and 3 indicates that VLP formation, and consequently VLP-incorporated minigenome transfer and expression, does not occur in the absence of M, F and G proteins. The results in lanes 4 through 15 shows that CAT levels varied in VLP infected samples depending on the concentration of M, F and G constructs used at transfection. Thus, the amount of M, F and G plasmids used at transfection had a bearing on the level of particle formation, and the consequent CAT reporter gene transfer and expression. CAT activity in the VLP infected reactions appeared optimal in the boxed lanes 7 and 8. Further analysis of CAT levels in the linear range (data not shown) demonstrated that optimal VLP formation was achieved with the ratios of M, F and G expression plasmids of 3∶1∶1 (lane 7).