Adeno-associated virus general transduction vectors: analysis of proviral structures.
We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (dl52-91) retains the AAV rep genes; the second (dl3-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%) contained proviruses which could be rescued (Res+) when the cells were superinfected with the appropriate helper viruses. Some produced high yields of viral DNA; other rescued at a 50-fold lower level. Most of the lines that were Res+ (79%) contained a tandem repeat of the AAV genome (2 to 20 copies) which was integrated randomly with respect to cellular DNA. Junctions between two consecutive AAV copies in a tandem array contained either one or two copies of the AAV terminal palindrome. Junctions between AAV and cellular sequences occurred predominantly at or within the AAV terminal repeat, but in some cases at internal AAV sequences. Two lines were seen that contained free episomal copies of AAV DNA. Res+ clones contained deleted proviruses or tandem repeats of a deleted genome. Occasionally, flanking cellular DNA was also amplified. There was no superinfection inhibition of AAV DNA integration. Our results suggest that AAV sequences are amplified by DNA replication either before or after integration and that the mechanism of replication is different from the one used during AAV lytic infections. In addition, we have described a new AAV general transduction vector, dl3-94, which provides the maximum amount of room for insertion of foreign DNA and integrates at a high frequency (80%).
McLaughlin
S K
SK
Department of Microbiology, University of Florida Medical School, Gainesville 32610.
Collis
P
P
Hermonat
P L
PL
Muzyczka
N
N
D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004261:DNA Replication; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D006378:Helper Viruses / Q000502:physiology; D006801:Humans; D010957:Plasmids; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D012641:Selection, Genetic; D014161:Transduction, Genetic; D014779:Virus Replication