Role of integrin alphaVbeta3 in the production of recombinant adenoviruses in HEK-293 cells.
Adenoviral infection is initiated by attachment of adenoviral fiber proteins to the CAR protein and subsequent internalization aided by alphaV -containing integrins, eg alphaVbeta3 and alphaVbeta5. To further understand the process of infection and assembly of recombinant adenoviral (rAd) vectors, we examined rAd production in HEK-293 cells and one of its subclones, clone D, isolated from the parental cells for high viral production. By flow cytometry, surface expression of integrin alphaVbeta3 by clone D cells was two-fold higher than by HEK-293 cells. However, clone D cells did not demonstrate greater translational efficiency or number of viral genome DNA copies shortly after rAd infection. Treating cells with inhibitors of integrin alphaVbeta3 reduced rAd production and transfecting HEK-293 cells with integrin alphaVbeta3 cDNAs increased rAd production. Subjecting cells to a sudden reduction in serum (10% to 0.1% FCS) for 5 days, clone D cells maintained 80% viability compared with 40% for HEK-293 cells. Further indication of survival signaling involvement was provided by Western blot analysis demonstrating p38 and p44/42 MAPKs were constitutively phosphorylated in HEK-293 cells. However, for clone D cells, p38 MAPK was phosphorylated only after rAd infection. The role of survival signaling mediated by integrin alphaVbeta3 in rAd production will be discussed.
Ling
W L W
WL
Department of Biotechnology Development, Schering-Plough Research Institute, Union, NJ 07083, USA.
Longley
R L
RL
Brassard
D L
DL
Armstrong
L
L
Schaefer
E J
EJ
D000074183:ATPases Associated with Diverse Cellular Activities; D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D002213:Capsid / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line / Q000821:virology*; D002470:Cell Survival; D002999:Clone Cells; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000821:virology; D008666:Metalloendopeptidases; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019030:Receptors, Vitronectin / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection / Q000379:methods; D048051:p38 Mitogen-Activated Protein Kinases
D015815:Cell Adhesion Molecules; D019030:Receptors, Vitronectin; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D008666:Metalloendopeptidases; C112889:SPG7 protein, human; D000074183:ATPases Associated with Diverse Cellular Activities