Chromosomal latency and expression at map unit 96 of a wild-type plus adeno-associated virus (AAV)/Neo vector and identification of p81, a new AAV transcriptional promoter.
OBJECTIVE
Human adeno-associated virus (AAV) is ubiquitous and known to establish latency by chromosomal integration. We have constructed a wild-type plus AAV vector, ins96-0.9Neo, containing the neomycin resistance gene open reading frame (Neo ORF) of 960 bases in length at map unit 96 of the virus. Ins96-0.9Neo was constructed in an unconventional manner in that the Neo ORF lacked a dedicated heterologous promoter. In this study, this wild-type plus AAV vector was to used to test AAV's packaging capacity and ability for chromosome 19 AAVS1 integration. However, when it was discovered that ins96-0.9Neo also transduced cells to G418 resistance, we also investigated the mechanism of Neo ORF expression in this vector.


METHODS
We investigated the ability of ins96-0.9Neo to produce virus at high titers and to retain the Neo sequences by Southern blot analysis. The ability of ins96 0.9Neo virus to transduce the Neo gene into cells was analyzed by colony formation under G418 selection, and the ability of ins96-0.9Neo to latently infect cells, including the AAVS1 region of chromosome 19, was investigated by a series of polymerase chain reaction (PCR) amplifications. Finally, the RNA expression of the Neo gene at map unit 96 was investigated by reverse transcriptase primer extension (RTPE) analyses with two different primers and by S1 nuclease protection.


RESULTS
High titers of the ins96-0.9Neo virus could be generated (10(9) infectious units [IU]/mL without concentration), the Neo gene was retained in the encapsidated viral genome, infection by this virus resulted in G418 resistance, and significant integration was taking place within the AAVS1 sequences of human chromosome 19 on transduction. Analysis of mRNA by RTPE using both primers and by the S1 nuclease protection assay mapped the 5' end of the Neo transcripts to approximately 700 bases upstream of the Neo ATG at map unit 81 (nt 3793-3813), thus identifying a new AAV promoter.


CONCLUSIONS
These data demonstrate that ins96-0.9Neo will be useful for studying wild-type AAV integration and suggest that such wild-type plus recombinant AAV vectors may be useful for human gene therapy. The advantages of using such wild-type plus AAV vectors over defective AAV vectors include the ease in production of recombinant virus and the ability for site-specific integration into chromosome 19. This study also uncovered a previously unknown AAV promoter, p81. This finding suggests that the as yet uncharacterized ORF (nt 3922-4388) located just downstream of this promoter is likely an expressed gene. Furthermore, these data support our earlier findings that the AAV virion can package >900 bases more than can the wild-type.
Hermonat
P L
PL
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Santin
A D
AD
De Greve
J
J
De Rijcke
M
M
Bishop
B M
BM
Han
L
L
Mane
M
M
Kokorina
N
N
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D004352:Drug Resistance, Microbial / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D009355:Neomycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014771:Virion; D016662:Virus Integration; D017735:Virus Latency; D014779:Virus Replication
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D012194:RNA-Directed DNA Polymerase; D009355:Neomycin