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Biomedical subjects

K I Berns

Publications and source records attributed to K I Berns.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1984

Recombination between simian virus 40 and adeno-associated virus: virion coinfection compared to DNA cotransfection.

Recombination between simian virus 40 (SV40) and adeno-associated virus (AAV) has been detected, by infectious center in situ plaque hybridization procedures, after both DNA contransfection and virion coinfection of monkey BSC-1 cells. The number of cells producing recombinants (1 in a 1000) was the same irrespective of the way in which the SV40 and AAV genomes were delivered to the cell, despite the fact that 5-10 times more cells were infected after virion coinfection. Several other dosage-response parameters of the recombination process consequent to virion coinfection were comparable to those after DNA cotransfection. The sole difference observed between the two infection systems was that the SV40/AAV recombinants formed after virion coinfection contained an inordinately high proportion of AAV terminal DNA sequences. By separating the SV40 and AAV infections in time, such that the AAV infection was delayed until after certain events in the SV40 cycle had taken place, an optimum phase for recombination in the SV40 cycle was identified. This phase occurs a few hours after infection, well before the onset of SV40 DNA replication and the synthesis of SV40-specific early proteins.

Animals↗
PubMed1984

Recovery of herpes simplex type 1 from the celiac ganglion after renal transplantation.

Herpes simplex type 1 was recovered from the celiac ganglion of a renal transplant patient who had esophageal lesions consistent with herpes simplex esophagitis as well as peptic ulcers requiring operation. We speculate that the virus got to the celiac ganglion via retrograde spread from the stomach and/or duodenum. Further studies are needed to determine the frequency of HSV involvement in visceral autonomic ganglia and whether by reactivation it could lead to subsequent gastrointestinal ulceration.

Esophagitis↗
PubMed1984

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.

Cell Line↗