A system for efficient generation of adenovirus protein IX-producing helper cell lines.
BACKGROUND
The adenovirus 14.3 kDa hexon-associated protein IX (pIX) functions in the viral capsid as 'cement' and assembles the hexons in stable groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type (wt) viruses, they can be propagated with the same kinetics and yields as the wt viruses. To facilitate 'pseudotyping' of adenoviral vectors we have set up an efficient system for the generation of pIX-producing helper cell lines.


METHODS
With a lentiviral pIX-expression cassette, monoclonal and polyclonal helper cell lines were generated, which express wt or modified pIX genes at levels equivalent to wt HAdV-5 infected cells. The incorporation efficiency into pIX gene deleted viruses was examined by Western analysis, immuno-affinity electron microscopy, and heat-stability assays.


RESULTS
Immuno-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX level in the helper cells was sufficient to generate heat-stable particles. Finally, the ratio between pIX and fiber was equivalent to that found in wt particles. The pIX-producing cell lines are very stable, demonstrating that pIX is not toxic to cells.


CONCLUSIONS
These data demonstrate that lentivirus vectors can be used for the establishment of pIX-complementing helper cell lines.
Vellinga
Jort
J
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Uil
Taco G
TG
de Vrij
Jeroen
J
Rabelink
Martijn J W E
MJ
Lindholm
Leif
L
Hoeben
Rob C
RC
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics*; D002461:Cell Line, Transformed; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D016253:Microscopy, Immunoelectron
D036022:Capsid Proteins