Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging.
Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination.
Maeda
Yasushi
Y
Department of Neurology, Kumamoto University, School of Medicine, 1-1-1 Honjo, Kumamoto, Kumamoto 860-0811, Japan. yasushi-maeda@fc.kuh.kumamoto-u.ac.jp
Kimura
En
E
Uchida
Yuji
Y
Nishida
Yasuto
Y
Yamashita
Satoshi
S
Arima
Toshiyuki
T
Uchino
Makoto
M
D000260:Adenoviruses, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D008969:Molecular Sequence Data; D017384:Sequence Deletion; D015398:Signal Transduction; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D004279:DNA, Viral; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases