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K I Berns

Publications and source records attributed to K I Berns.

At least 19 recordsLinked to original sources
PubMed1992

In vitro replication of adeno-associated virus DNA.

An in vitro assay for adeno-associated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the plasmid by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I digestion. The following results were obtained. (i) Only extracts of cells coinfected with AAV and adenovirus replicated the excised insert. (ii) Density label experiments showed semiconservative replication. (iii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.

Adenoviruses, Human↗
PubMed1992

Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination.

The human parvovirus, adeno-associated virus (AAV), has been shown to integrate preferentially into human chromosome 19 q13.3-qter. The human target sequence for AAV integration (AAVS1) was cloned and sequenced. By analysis of the proviral junctions it was determined that integration of the AAV DNA occurred via a non-homologous recombination pathway although there were either four or five identical nucleotides at the junctions. Integration was a multistep, concerted process that resulted in cellular sequence rearrangements. The sequence of the integration locus was analyzed for possible recombination signals. Direct repeats at a much greater than random occurrence were found distributed non-uniformly throughout the AAVS1 sequence. A CpG island containing transcription factor binding site elements is suggestive of a TATA-less promoter. Evidence for transcriptional activity was provided by PCR amplification of reverse transcribed RNA.

Base Sequence↗
PubMed1991

In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome.

In an in vitro simian virus 40 (SV40) DNA replication assay, we have observed excision of a hybrid adeno-associated virus (AAV)/SV40 insert from a plasmid construct. The excision was dependent on the presence of the palindromic AAV terminal repeat and greatly enhanced by the addition of the SV40 T antigen to the reaction. Analysis of the excision product supports a model in which the palindromic terminal sequences of AAV form a cruciform structure (equivalent to a Holliday recombination intermediate), which is cleaved and resealed so that the excision products are linear duplex pBR322 and linear duplex AAV/SV40 insert. Both the excised linear duplex pBR322 and the excised linear duplex AAV/SV40 insert have each terminus covalently crosslinked by one copy of the palindromic region of the AAV terminal repeat region folded on itself. The excision process may be a model system for cellular homologous recombination. The process as observed was either concomitant with or subsequent to DNA replication.

Adenoviruses, Human↗
PubMed1990

Site-specific integration by adeno-associated virus.

Cellular sequences flanking integrated copies of the adeno-associated virus (AAV) genome were isolated from a latently infected clonal human cell line and used to probe genomic blots derived from an additional 21 independently derived clones of human cells latently infected with AAV. In genomic blots of uninfected human cell lines and of primary human tissue, each flanking-sequence probe hybridized to unique bands, but in 15 of the 22 latently infected clones the flanking sequences hybridized not only to the original fragments but also to a total of 36 additional species. AAV probes also hybridized to 22 of these new bands, representing 11 of the 15 positive clones, but never to the fragment characteristic of uninfected cell DNA. From these data we conclude that the AAV genome preferentially integrates into a specific region of the cellular genome. We have determined that the integration site is unique to chromosome 19 by somatic cell hybrid mapping, and this sequence has been isolated from uninfected human DNA.

Cell Line↗
PubMed1990

Parvovirus replication.

The members of the family Parvoviridae are among the smallest of the DNA viruses, with a linear single-stranded genome of about 5 kilobases. Currently the family is divided into three genera, two of which contain viruses of vertebrates and a third containing insect viruses. This review concentrates on the vertebrate viruses, with emphasis on recent advances in our insights into the molecular biology of viral replication. Traditionally the vertebrate viruses have been distinguished by the presence or absence of a requirement for a coinfection with a helper virus before productive infection can occur, hence the notion that the dependoviruses (adeno-associated viruses [AAV]) are defective. Recent data would suggest that not only is there a great deal of structural and genetic organizational similarity between the two types of vertebrate viruses, but also there is significant similarity in the molecular biology of productive replication. What differs is the physiological condition of the host cell that renders it permissive. Healthy dividing cells are permissive for productive replication by autonomous parvoviruses; such cells result in latent infection by dependoviruses. For a cell to become permissive for productive AAV replication, it must have been exposed to toxic conditions which activate a latent AAV genome. Such conditions can be caused by helper-virus infection or exposure to physical (UV light) or chemical (some carcinogens) agents. In this paper the molecular biology of replication is reviewed, with special emphasis on the role of the host and the consequences of viral infection for the host.

Animals↗
PubMed1989

Interactions between the termini of adeno-associated virus DNA.

The adeno-associated virus (AAV) genome is a linear, single polynucleotide chain with inverted terminal repeats of 145 bases. In order to test whether the terminal repeats at opposite ends of the genome have to be able to completely base-pair during DNA replication, we have created chimeric genomes in which an 11 base symmetrical sequence has been deleted from the terminal repeat at one end of the genome and replaced by a different 12 base symmetrical sequence. We have used these chimeric constructs either as a duplex insert in pBR322 or as purified duplex virion DNA to transfect adenovirus-infected HeLa cells. When chimeric duplex virion DNA was used, all of the progeny virions obtained after two cell passages contained DNA with wild-type sequences in both terminal repeats. When plasmid clones were used, the structure of virion DNA depended on the original orientation. If the mutant terminal repeat was originally at the right end of the genome (terminus of genetic map), all progeny terminal repeat sequences were again wild-type. However, if the original construct contained the mutant sequence in the left terminal repeat, the majority of progeny molecules were parental in type (i.e. mutant left and wild-type right terminal repeat). We conclude (1) although the terminal repeats at opposite ends of the genome may interact during DNA replication, it is not necessary that they be perfectly complementary. (2) In direct competition, the wild-type sequence displays an advantage over the mutant allele. (3) In a plasmid clone, the terminal repeat on the left end of the genome is at an advantage in a competitive situation. We note that the left terminal repeat is adjacent to a transcriptional promoter.

Base Sequence↗
PubMed1989

Organization of adeno-associated virus DNA in latently infected Detroit 6 cells.

The DNA of a human dependovirus, adeno-associated virus (AAV), integrates into the cellular genome under conditions nonpermissive for viral DNA replication. The AAV DNA can be rescued from the cellular genome by superinfection with a helper virus, e.g., adenovirus. To characterize the organization of the proviral DNA in greater detail, we isolated three proviral subfragments from a latently infected human cell line. Our findings, based on sequence analysis, restriction enzyme mapping, and genomic blots, demonstrate that the viral terminal repeats (trs) are present at or near the cellular/viral DNA junctions, but significant deletions of the tr sequences have occurred. One of the proviral clones has extensive rearrangements which apparently occurred by nonhomologous recombination. The tail-to-tail arrangement of one of the proviral clones is consistent with limited DNA replication prior to integration by a mechanism similar to that seen in a productive infection. Although there are BamH1 fragments in the cell line we have characterized, which are the right size to have come from a head-to-tail tandem repeat, critical double digests show no evidence for the presence of a head-to-tail tandem repeat of wild-type proviral DNA. Use of probes derived from the flanking cellular DNA enabled us to determine that in this cell line the viral DNA had integrated into a site composed of nonrepetitive sequences.

Base Sequence↗
PubMed1989

Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein.

The leftward two promoters of the adeno-associated virus (AAV) 2 genome were fused to reporter genes, and the constructs were used to transfect HeLa cells. The promoters functioned constitutively but were repressed in trans by the AAV rep gene product(s). The repression was relieved by adenovirus infection. Evidence which indicated an enhancer function for the inverted terminal repeat of the AAV-2 genome was also obtained.

Chloramphenicol O-Acetyltransferase↗
PubMed1988

Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat.

The defective parvovirus, adeno-associated virus (AAV), contains a single-stranded DNA genome of 4681 bases with inverted terminal repeats of 145 bases. The distal 125 bases of the repeat are palindromic allowing a hairpin to form for initiation of DNA synthesis. The palindromic region contains three palindromes, two smaller internal palindromes flanked by a larger palindrome, which allow the hairpinned DNA to assume a T-shaped conformation during DNA replication. Deletion of an internal palindrome forming one of the crossarms of the T results in the inability of the AAV genome to be rescued from plasmid sequences and replicated. Restoration of the crossarm sequences with DNA that differs in primary sequence but maintains the symmetry of the palindrome results in viable AAV and propagation of the mutant sequences. In this paper we report further studies on the nature of mutants made within the crossarm of the T. Two types of substitution mutants were analyzed. Symmetrical sequence substitution mutants were viable as previously reported. An analysis of the kinetics of AAV DNA accumulation showed that the symmetrical sequence substitution mutants were indistinguishable from wild-type AAV. This was true if the AAV DNA was introduced into the cells either as plasmid DNA or as DNA extracted from virions. In contrast, intermolecular competition experiments showed either a dominance of the wild-type sequence or codominance of both sequences when both alleles were cotransfected into helper virus-infected cells. A preference for the wild-type sequence may also exist but is not required for efficient AAV replication. The second type of mutation studied was an asymmetrical sequence substitution mutant. This mutant was replicated but at a level too low to be propagated. These data suggest that symmetry is required in the internal palindromic region, presumably for the formation of the crossarm structure in the T-shape.

Cloning, Molecular↗
PubMed1988

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.

Adenoviridae↗
PubMed1987

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

In this paper we report that adeno-associated virus (AAV) genomes inhibit stable transformation by several dominant selectable marker genes upon cotransfection into mouse tissue culture cells. Cotransfection of AAV genomes also inhibited the expression of pSV2cat in transient assays. In both cases, the inhibitory effect was independent of AAV DNA replication but required the AAV p5 and p19 genes, which encode proteins required for AAV DNA replication and regulation of AAV gene expression. Finally, addition of a cloned E4 gene in the transfection experiments partially blocked the AAV-mediated inhibitory activities.

Acetyltransferases↗
PubMed1986

Positive and negative autoregulation of the adeno-associated virus type 2 genome.

The defective human parvovirus, adeno-associated virus (AAV), requires multiple functions provided by a coinfecting helper virus for viral replication. In addition, it has recently been shown that at least one AAV gene is also required for AAV DNA replication. In this paper, we investigate the autoregulation of the AAV genome by analyzing the expression of mutant AAV genomes upon transfection into adenovirus-infected human cells. Evidence is presented which indicates that the AAV genome regulates its own gene expression in at least two ways. First, either the AAV p5 gene or both the p5 and p19 genes appear to encode a trans activator of AAV transcription. Frameshift mutations within the p5 or p19 gene severely inhibited the synthesis and accumulation of all AAV transcripts. The defective accumulation of transcripts could be complemented in trans, in a manner independent of DNA replication, by cotransfection with a capsid deletion mutant. Second, evidence is presented which suggests that the p5 and p19 genes contain negative cis-active regulatory elements. Deletion of sequences within the p5 and p19 genes enhanced the accumulation of the p5 transcript in cis upon complementation with an AAV capsid deletion mutant, whereas certain deletions enhanced p40 RNA accumulation in the absence of trans activation by the p5 gene.

DNA Replication↗
PubMed1985

Structure of simian virus 40-adeno-associated virus recombinant genomes.

The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids.

Base Sequence↗