The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVSI). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA- The life cycle of the human parvovirus, adeno-associated virus (AAV), is characterized by persistent infection (1). This is manifest in cell culture by the inability of AAV to undergo productive infection in the absence of a concomitant infection by a helper virus, such as adenovirus (2, 3) or herpesvirus (4, 5), or exposure of the host cell to genotoxic conditions able to induce a stress response (6-8). Although all nuclear DNA viruses commonly cause persistent infections in the intact host, the peculiar properties of AAV have made it a particularly useful model system for study of the molecular mechanisms underlying the establishment of a latent infection. Infection of continuous lines of human cells in culture by AAV at a multiplicity of infection of 20-200 in the absence of a helper virus leads to a high percentage of cells containing the viral genome in a latent chromosomally integrated state (9-11). Activation of the latent AAV genome is readily accomplished by superinfection of the latently infected cell with a helper virus (12).The AAV genome is a linear, single-stranded DNA of 4.7 kb, which contains two major open reading frames; regulatory proteins are encoded in the left half of the genome, and structural proteins are encoded in the right half (13). Our current model of the AAV life cycle has the following elements. (i) When AAV infects a healthy, dividing cell, there is a very limited expression of Rep 68/78, which has three biological consequences. Further AAV gene expression is repressed; to a very great extent, AAV DNA replication is inhibited; and integration of AAV DNA into the host cell genome occurs. All three of these are directly dependent on Rep 68/78. (ii) Infection of a latently infected cell by a helper virus leads to activation of the integrated AAV genome, rescue from the integrated state, DNA replication, and production of progeny virions. Rep 68/78 is required during this process for gene expression, DNA replication, and rescue from the integrated state. Thus, the phenotype of Rep 68/78 expression is directly dependent on the physiological state of the host cell. When the cell is healthy, the AAV latent state is maintained; when the cell is stressed, the viral genome is activated to allow infection of a new host.Our goal has been to understand the molecular mechanisms underlying the phenomenon of AAV latency. Although any exogenous DNA introduced into a mammalian cell can recombine in a nonhomologous manner with the host DNA, the AAV genome is the only example of an exogenous DNA that integrates at a specific site (19ql3.3-qter) in the human genome (16)(17)(18)(19). Our current understanding of this p...

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVSI). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals withinAAVSl required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It Complementation studies with temperature-sensitive mutants of the tumor-causing simian virus 40 (SV40) indicate that it may have as few as three primary gene products (1). Only one of these, the A gene product, has been directly linked to the process of virus-induced neoplastic transformation (2-5). Cells in the early phase of productive lytic infection and virus-transformed cells exhibit some common features. In each case, a species of virus-specific RNA is induced, which hybridizes to the same strand of SV40 DNA (6, 7) and to the same fragments of SV40 DNA generated by restriction endonucleases (8, 9). At least three virus-associated antigens, T, U, and TSTA, have been detected both early in lytic infection and in transformed cells (10). The T-antigen has so far been characterized primarily immunologically, employing sera from animals bearing SV40-induced tumors (11-13). T-antigen has been partially purified (14-16) and is now considered to be a DNA-binding protein (17)(18)(19)(20) with the molecular weight of the denatured polypeptide estimated to be from 70,000 (21) to 100,000 (22).Comparison of the properties of the T-antigen after infection with either wild-type or A-mutant virus at the restrictive temperature has supplied evidence that this antigen (or a part thereof) is encoded by viral DNA (22-24). To demonstrate directly that the information for the SV40 T-antigen is encoded in the viral genome, and to study its expression further, we have investigated the cell-free translation of early SV40 mRNA.We report that SV40 early mRNA from infected cells, and SV40 cRNA transcripts made in vitro, direct the synthesis in the wheat-germ cell-free system of polypeptides that can be specifically immunoprecipitated by antiserum to SV40 Tantigen. The products directed by "in vivo" mRNA and synthetic cRNA differ, however, in their electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide gels. The former products correspond to the T-antigen immunoprecipitated from extracts of infected cells and the largest of these has an estimated molecular weight of 90,000. A preliminary description of these findings has been given previously (25). Antiserum to SV40 T-antigen was obtained from Flow Laboratories. Goat anti-hamster serum was a gift from M. Fogel. MATERIALS AND METHODSCells and Viruses. Plaque-purified SV40 (strain 777) was grown in the BS-C-1 line of African green monkey cells as previously described (26).Cell Extracts. Cultures (107 cells) of uninfected or SV40-infected cells (multiplicity of infection = 2 X 109 plaqueforming units/ml) were labeled from 47 to 48 hr post infection with [35S]methionine, (20 MCi/ml of methionine-free medium). After...