Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions.

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.     

A novel terminal resolution-like site in the adeno-associated virus type 2 genome.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.     

Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.     

Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.     

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.     

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.     

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.     

Four-dimensional visualization of the simultaneous activity of alternative adeno-associated virus replication origins

The adeno-associated virus (AAV) inverted terminal repeats (ITRs) contain the AAV Rep protein-binding site (RBS) and the terminal resolution site (TRS), which together act as a minimal origin of DNA replication. The AAV p5 promoter also contains an RBS, which is involved in Rep-mediated regulation of promoter activity, as well as a functional TRS, and origin activity of these signals has in fact been demonstrated previously in the presence of adenovirus helper functions. Here, we show that in the presence of herpes simplex virus type 1 (HSV-1) and AAV Rep protein, p5 promoter-bearing plasmids are efficiently amplified to form large head-to-tail concatemers, which are readily packaged in HSV-1 virions if an HSV-1 DNA-packaging/cleavage signal is provided in cis. We also demonstrate simultaneous and independent replication from the two alternative AAV replication origins, p5 and ITR, on the single-cell level using multicolor-fluorescence live imaging, a finding which raises the possibility that both origins may contribute to the AAV life cycle. Furthermore, we assess the differential affinities of Rep for the two different replication origins, p5 and ITR, both in vitro and in live cells and identify this as a potential mechanism to control the replicative and promoter activities of p5.     

Encapsidation of adenovirus 16 DNA is directed by a small DNA sequence at the left end of the genome

We have identified a DNA sequence in adenovirus type 16 which contains recognition signals for encapsidation of the viral DNA. The sequence acts in cis to direct the encapsidation of DNA from the end of the viral genome where it is located. The sequence is normally contained in the first 390–400 bp of the left end of the genome. The location was determined by analyzing a series of spontaneous mutants of Ad16 which carried reduplications of 200 to >500 bp of left end sequences at the right end of the genome, thus giving rise to enlarged inverted terminal repetitions (ITR). In plaque-purified (PP) Ad16 prototype virus the subgenomic DNA found in incomplete virus particles exclusively represents left end sequences. When the reduplication mutants were analyzed, we found that a reduplication of about 390 bp enabled subgenomic DNA molecules containing the right end to be encapsidated into incomplete particles as well. A reduplication of about 290 bp, however, did not allow subgenomic DNA containing the right end to be encapsidated. The difference in encapsidation described could not be attributed to an asymetric DNA replication in the mutants, since subgenomic DNA originating from both ends of the genome was produced in equal amounts in the infected cells. We conclude that an essential part of the encapsidation sequence must be located between 290 and 390 bp from the left end of the Ad16 genome.     

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%).     

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.     

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.     

Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.     

Analysis of the Effects of Charge Cluster Mutations in Adeno-Associated Virus Rep68 Protein In Vitro

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Δ) expressed in Escherichia coli cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.     

Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells.

A clone of human cells (Detroit 6) latently infected by adeno-associated virus (AAV) has been characterized with regard to the status of the viral DNA. In both early (9 to 10) and late (118) passages of the clone, AAV-DNA was recombined with host DNA, at least in some cases as a head-to-tail tandem repeat, via the terminal sequences of the viral genome. However, it was not possible to distinguish between integration into chromosomal DNA and very large plasmids (< 20 x 10(6) molecular weight) which contain both viral and cellular DNA sequences. Although evidence for some modifications of the viral sequence was obtained, most of the integrated sequences appeared to be intact. In some cases sequences of undetermined origin separated adjacent copies of the viral genome. Free copies of the AAV genome were detectable in late passage cells, but not in early passage cells. The orientation of nucleotide sequences present in the free AAV DNA from late passage cells was indistinguishable from that of virion DNA. With the notable exception, the organization of the integrated AAV sequences as determined by restriction enzyme digestion remained constant with continued passage. Digestion with SmaI, which cleaves within the palindromic region of the terminal repetition in AAV DNA, produced reproducibly different patterns when early and late passage DNAs were compared. Several models for rescue of free copies of the genome from the integrated DNA are possible, all of which involve the terminal repetition.