Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

Isolation of the proviral coding strand of avian myeloblastosis virus from leukemic chicken myeloblast by affinity chromatography.

The coding strand of the integrated proviral DNA of avian myeloblastosis virus (AMV) was isolated from the DNA of leukemic chicken myeloblast. The three-step isolation procedure employed a combination of affinity chromatography with Sepharose-linked RNA, nucleic acid hybridization, and hydroxypatite chromatography techniques. At each step of purification the product was analyzed for the enrichment of AMV coding strand by hybridization with AMV RNA. The final product was the coding strand of the AMV DNA (90% pure). These results show that such a procedure can be used for the isolation and analysis of a specific structural gens of eukaryotic cells.     

Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication

In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.     

Single-strandedness of the majority of DNA sequences complementary to mRNA-coding sites isolated from rat hepatoma tissue cultured cells

Single-stranded DNA (ssDNA), separated from bulk double-stranded DNA (dsDNA) of HTC by an improved method of hydroxyapatite chromatography, exhibited the same characteristics as ssDNA previously found in various cell species. It amounted to 1.5–2% of the total nuclear DNA. Only 24–26% could be self-reassociated, but the greatest part hybridized to non-repetitious DNA fraction and about 30% hybridized to homologous mRNA.Other results tend to prove that the complementary sequences of HTC-ssDNA probably consist of non-base-paired segments attached to double helical regions of dsDNA. In effect, after hydroxyapatite chromatography, a small portion of HTC-dsDNA (2–3%) was found to be rapidly digestible by S1 nuclease and this limited digestion was sufficient to reduce markedly the hybridization rates of dsDNA with both DNA and cell-free synthesised cDNA copies of polyadenylated RNAs. Furthermore, these ³H-cDNA copies could not be annealed to ssDNA under conditions that allowed their reassociation with total nuclear DNA. These findings complete the demonstration that the greatest part of ssDNA appears to be formed via selective nicks, probably enzymatic, in the coding strand of actively transcribed DNA regions.     

Genetic Relatedness Studies with Adenovirus-associated Viruses

Adenovirus-associated viruses (AAV) contain double-stranded deoxyribonucleic acid (DNA). DNA from each of the four AAV serotypes was used as template for the in vitro synthesis of complementary (3)H-ribonucleic acids(RNA). An estimation of genetic interrelatedness was made on the basis of hybridization reactions between synthetic AAV RNA and AAV DNA. Heterologous reactions were 27 to 37% of homologous reactions, suggesting that the AAV serotypes are related to about the same extent. AAV-1 synthetic RNA was also reacted with DNA from helper adenovirus types 2, 7, and SV15. Very low levels of RNA binding were observed, but it is not likely that these reactions represent AAV-adenovirus genetic relatedness.     

Role of the herpes simplex virus helicase-primase complex during adeno-associated virus DNA replication

A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.     

Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins

Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.     

A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication.

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.     

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.     

Herpes Simplex Virus Types 1 and 2 Completely Help Adenovirus-Associated Virus Replication

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.