Substitution of adeno-associated virus Rep protein binding and nicking sites with human chromosome 19 sequences.

BACKGROUND: Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated AAVS1 (also known as MBS85). Integration at AAVS1 requires the AAV2 replication (Rep) proteins and a DNA sequence within AAVS1 containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or trs). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced trs, but the sequences differ from those in AAVS1. These ITR sequences are required for replication and packaging. RESULTS: In this study we demonstrate that the AAVS1 RRS and trs can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of AAVS1 DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at AAVS1, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to AAVS1 sequences. CONCLUSIONS: The ability of these AAVS1 sequences to substitute for the AAV2 RRS and trs provides indirect evidence that the stable secondary structure encompassing the trs is part of the AAV2 packaging signal.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.

BACKGROUND: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. RESULTS: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. CONCLUSION: AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Analysis of DNA binding by a eubacterial zinc finger transcription factor.

The Serratia marcescens NucC protein is structurally and functionally homologous to the P2 Ogr family of eubacterial zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. These activators exhibit site-specific binding to a conserved DNA sequence, TGT-N(3)-R-N(4)-Y-N(3)-aCA, that is located upstream of NucC-dependent S. marcescens promoters and the late promoters of P2-related phages. In this report we describe the interactions of NucC with the P2 FETUD late operon promoter P(F). NucC is shown to bind P(F) as a tetramer and to make 12 symmetrical contacts to the DNA phosphodiester backbone. The backbone contacts are centered on the TGT-N(3)-R-N(4)-Y-N(3)-aCA motif. Major groove base contacts can be seen at most positions within the approximately 24-bp binding site. Minor groove contacts map to adjacent positions in the downstream half of the binding site, which corresponds to the area in which the DNA also appears to be bent by NucC binding. NucC binding provides a new example of protein-DNA interaction that is strikingly different from the DNA binding demonstrated for eukaryotic zinc-finger transcription factors.

Preferential integration of adeno-associated virus type 2 into a polypyrimidine/polypurine-rich region within AAVS1.

Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-end-joining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.