Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

Effects of adeno-associated virus DNA hairpin structure on recombination

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.     

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.     

转染腺相关病毒对骨髓间充质干细胞分化潜能的影响

目的:探讨转染腺相关病毒对骨髓间充质干细胞分化潜能的影响.方法:运用密度梯度离心法分离骨髓间充质干细胞;将pAAV-GFP、pAAV-RC、pHelper用磷酸钙法共转染HEK-293细胞,得到rAAV-GFP,转染骨髓间充质干细胞.进行成骨诱导分化,观察rAAV-GFP对骨髓间充质干细胞分化潜能的影响.结果:与未转染rAAV-GFP的间充质干细胞相比较,转染rAAV-GFP后,骨髓间充质干细胞分化潜能未见改变,均表现为细胞浆蓝紫色,核周明显.结论:转染腺相关病毒对骨髓间充质干细胞分化潜能未见明显影响,为基因修饰腺相关病毒转染骨髓间充质干细胞进行体内移植提供了实验基础.     

Spatial and temporal organization of adeno-associated virus DNA replication in live cells

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.     

Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells.

The mechanism of adeno-associated virus (AAV) DNA replication was characterized both genetically and biochemically. In this study, we used monoclonal and polyclonal antibodies to examine the AAV p5 (Rep78 and Rep68) and p19 (Rep52 and Rep40) proteins in infected cells. By overexpressing a truncated Rep78 protein in Escherichia coli, we obtained monoclonal antibody anti-78/68, which is specific for the p5 Rep proteins, and monoclonal antibody anti-52/40, which recognized both the p5 and p19 Rep proteins. In single-fluorochrome indirect immunofluorescence labeling experiments, the viral Rep proteins were localized in distinct intranuclear foci. Analysis of AAV proteins by double-fluorochrome indirect immunofluorescence experiments demonstrated that (i) all four AAV Rep proteins occupied the same intranuclear compartments and (ii) the Rep and capsid proteins colocalized in the nuclei of infected cells. These results suggest that replication centers similar to those established by other viruses exist for AAV. These reagents should provide a useful tool for further delineation of the mechanism of AAV replication in vitro.     

Arrangement of nucleotide sequences in adeno-associated virus DNA

There are two types of adeno-associated virus virions which contain complementary single-stranded DNA genomes of about 1.4 × 10⁶ daltons. The purified complementary single polynucleotide chains anneal to form duplex linear monomers, circular monomers, and linear dimers, in addition to other less well-defined structures, as identified by sedimentation and electron microscopy. All duplex species are formed by linear single polynucleotide chains of unit length, thus duplex circles and linear dimers are assumed to be held together by relatively short overlapping hydrogen-bonded regions. The initial linear monomers present after annealing the complementary single strands do not form duplex circles or oligomers when re-exposed to annealing conditions, but DNA which sediments as linear monomers after heating linear dimers at a temperature from 7 to 25 deg. C below the T_m of duplex AAV² DNA does re-form oligomers and circles when exposed to annealing conditions. Denaturation and reannealing of any duplex species leads to the formation of all forms, indicating that the over-all single strand composition of all species is equivalent. Disruption of duplex circles by limited exonucleolytic digestion using either 3′ or 5′ exonucleases leads to the conclusion that the overlap region may have either 3′ or 5′ termini and that the overlap region represents less than 6% of the length of the genome. Exonucleolytic digestion of linear monomers to the extent of 50% leaves polynucleotide chains which cannot reanneal after denaturation, thus AAV DNA is not randomly circularly permuted. Duplex linear monomers which do not form circles when exposed to annealing conditions do form duplex circles after 1% exonuclease III digestion. A model consistent with these data is one in which the linear single polynucleotide chains present in AAV virions consist of two or more permutations. All of these chains contain terminal repetitions and their start points occur within a limited region, representing < 6% of the length of the genome. According to this interpretation AAV DNA would exist within the virion as a linear single polynucleotide chain or is cleaved at a few specific sites during extraction.     

Sequence organization of feline leukemia virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.     

Study of the fine structure of adeno-associated virus DNA with bacterial restriction endonucleases.

A physical map of the adeno-associated virus type 2 genome has been constructed on the basis of the five fragments produced by the restriction endonucleases HindII + III from Hemophilus influenzae. There are three endo R-HindII cleavage sites and one endo R-HindIII site. Evidence has been obtained to support the existence of two nucleotide sequence permutations in adeno-associated virus DNA, the start points of which have been estimated to be separated by 1% of the genome. The three cleavage fragments produced by endo R-Eco RI have been ordered and oriented with respect to the endo R-HindII + III cleavage map.     

Intermediates of adeno-associated virus DNA replication in vitro.

Intermediates of adeno-associated virus type 2 (AAV) DNA replication in an in vitro assay have been characterized. The assay involves rescue and replication of an AAV insert in pBR322. Intermediates were shown to be duplex molecules in which at least one terminus was in the extended configuration, in contrast to the hairpinned ends seen after rescue in the absence of AAV DNA replication. Also present were linear double-stranded dimers, which were characterized as either head-to-head or tail-to-tail tandems; no head-to-tail dimers were detected. The results are in accord with the current model of AAV DNA replication.     

腺相关病毒的衣壳装配和DNA衣壳化机制

重组腺相关病毒载体 (rAAV) 是基因治疗临床应用载体的选择之一。在简述野生型AAV基因组结构和复制机制的基础上,阐述了AAV包装过程中两个主要事件：衣壳蛋白的装配和基因组DNA的衣壳化。虽然对AAV的包装机制总体上已有一定的认识,但其详细的分子机制、构效关系仍需完善和充实。AAV病毒本身相关机制的深入研究有助于改善rAAV载体的制备技术,促进rAAV基因药物研发。     

In vitro replication of adeno-associated virus DNA.

The study of eukaryotic viral DNA replication in vitro has led to the identification of cellular enzymes involved in DNA replication. Adeno-associated virus (AAV) is distinct from previously reported systems in that it is believed to replicate entirely by leading-strand DNA synthesis and requires coinfection with adenovirus to establish completely permissive replication. In previous work, we demonstrated that two of the AAV nonstructural proteins, Rep78 and -68, are site-specific endonucleases and DNA helicases that are capable of resolving covalently closed AAV termini, a key step in AAV DNA replication. We have now cloned the AAV nonstructural proteins Rep78, Rep68, and Rep52 in the baculovirus expression system. Using the baculovirus-expressed proteins, we have developed an efficient in vitro AAV DNA replication system which mimics the in vivo behavior of AAV in every respect. With no-end AAV DNA as the starting substrate, the reaction required an adenovirus-infected cell extract and the presence of either Rep78 or Rep68. Rep52, as expected, did not support DNA replication. A mutant in the AAV terminal resolution site (trs) was defective for DNA replication in the in vitro assay. Little, if any, product was formed in the absence of the adenovirus-infected HeLa cell extract. In general, uninfected HeLa extracts were less efficient in supporting AAV DNA replication than adenovirus-infected extracts. Thus, the requirement for adenovirus infection in vivo was partially duplicated in vitro. The reduced ability of uninfected HeLa extracts to support complete DNA replication was not due to a defect in terminal resolution but rather to a defect in the reinitiation reaction or in elongation. Rep78 produced a characteristic monomer-dimer pattern of replicative intermediates, but surprisingly, Rep68 produced little, if any, dimer replicative form. The reaction had a significant lag (30 min) before incorporation of 32P-deoxynucleoside triphosphate could be detected in DpnI-resistant monomer replicative form and was linear for at least 4 h after the lag. The rate of incorporation in the reaction was comparable to that in the simian virus 40 in vitro system. Replication of the complete AAV DNA molecule was demonstrated by the following criteria. (i) Most of the monomer and dimer product DNAs were completely resistant to digestion with DpnI. (ii) Virtually all of the starting substrate was converted to heavy-light or heavy-heavy product DNA in the presence of bromo-dUTP when examined on CsCl density gradients.(ABSTRACT TRUNCATED AT 400 WORDS)