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B J Carter

Publications and source records attributed to B J Carter.

At least 91 records · Page 5Linked to original sources
PubMed1984

Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions.

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

Autoradiography↗
PubMed1984

Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells.

Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes. We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328. Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom. The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure. Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes. The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.

Base Sequence↗
PubMed1984

Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function.

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.

Capsid↗
PubMed1984

A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase.

We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

Acetyltransferases↗
PubMed1984

Analysis of two potential shuttle vectors containing herpes simplex virus defective DNA.

Two potential shuttle vectors which contained the identical herpes simplex virus type 1 (HSV-1) defective particle DNA (dDNA), but prokaryotic DNA of different origins, were examined for their stability when propagated in eukaryotic cells, and for their efficiency as shuttle vectors. Each chimeric molecule contained a 9.5 kilobase-pair (kb) EcoRI fragment (HSV12-7) representing a single unit of a class I HSV-1 dDNA. This dDNA was cloned into the bacteriophage lambda (lambda) vector lambda gtWES X lambda B' to create a 45.3 kb chimeric molecule (lambda gtWES::12-7), and into the plasmid vector pBR325, resulting in a 15.5 kb recombinant DNA molecule (pBR325::12-7). Each of these DNA molecules was transfected independently into African green monkey kidney cells which were then infected with wild-type HSV-1 helper virus. Both chimeric molecules were replicated and packaged into HSV-1 virions. However, regions of the lambda gtWES::12-7 chimeric DNA were rapidly deleted and rearranged, whereas the plasmid/HSV-1 DNA molecules were less rearranged. No intact lambda gtWES::12-7 DNA was recovered from HSV-1 virions as detected by infectivity of in vitro packaged DNA. However, pBR325::12-7 DNA isolated from HSV-1 virions was able to transform E. coli to ampicillin resistance. These results suggest additional considerations when designing single units of HSV-1 dDNA for use as vectors to accommodate large fragments of DNA.

Animals↗
PubMed1983

Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication.

We have analyzed the ability of an adenovirus type 2 mutant, Ad2dl807, to support replication of adeno-associated virus (AAV). This mutant has a deletion extending from early region 3 through the late fiber gene and into early region 4. Since AAV growth does not require Ad early region 3 or the fiber gene, the Ad2dl807 mutant allows analysis of the function of Ad early region 4 in AAV growth. As determined by assay of AAV DNA, RNA, and protein synthesis as well as the number of cells producing AAV and the yield of infectious AAV particles, growth of AAV with the dl807 mutant is decreased 10- to 20-fold. The residual AAV growth is probably due to the presence of a low level of contaminating viable Ad virions in the dl807 preparations. These observations indicate that an Ad early region 4 function is required at a very early stage of AAV DNA replication to allow amplification of duplex replicating (RF) DNA. Thus growth of AAV offers an independent probe for adenovirus early region 4 functions.

Adenoviruses, Human↗
PubMed1983

Characterization of the herpes simplex virus type 1 glycoprotein D mRNA and expression of this protein in Xenopus oocytes.

We have identified and characterized a 3.0 kilobase (kb) mRNA containing coding sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene. The synthesis of this 3.0 kb mRNA was unaffected by the presence of cytosine arabinoside, but was made in greatly reduced amounts in cells infected with HSV-1 in the presence of cycloheximide: it was, therefore, classified as an early mRNA. By nuclease protection experiments, it was found that the 3.0 kb mRNA is unspliced and, further, that it is 3' co-terminal with a smaller 1.6 kb early mRNA which is transcribed from a DNA sequence 3' to the gD coding sequence. We describe the use of the Xenopus laevis oocyte system to produce HSV-1 gD in vitro. Oocytes injected with mRNA isolated from HSV-1-infected Vero cells synthesized gD, which was identified by immunoprecipitation. Injection of a plasmid clone containing the HSV-1 BamHI J fragment (0.89 to 0.93 map units) into the nuclei of Xenopus oocytes also resulted in synthesis of gD.

Animals↗
PubMed1983

A lambda library of Herpes simplex virus type 1 (KOS) DNA fragments obtained by partial digestion with Sau3A.

Large fragments of Herpes simplex virus type 1 (HSV-1, strain KOS) DNA were produced by partial cleavage with Sau3A and inserted into a phage lambda BamHI vector. Recombinant phage (lambda KOS) DNA molecules were isolated and characterised. The final collection of phage recombinants contains partially overlapping inserts, which represent most of the HSV-1 genome. Restriction enzyme analysis of many independent clones containing Us sequences revealed sequence polymorphism in two specific regions.

Bacteriophage lambda↗
PubMed1983

Cloning of infectious adeno-associated virus genomes in bacterial plasmids.

We describe the construction of two Escherichia coli hybrid plasmids, each of which contains the entire 4.7-kb DNA genome of the human parvovirus, adeno-associated virus (AAV) type 2. Because the AAV genome was inserted into the plasmid DNA using BglII linkers the entire virus genome can be recovered by in vitro cleavage of the purified recombinant plasmid. Transfection of these recombinant DNAs into an adenovirus-transformed human cell line in the presence of helper adenovirus resulted in efficient rescue and replication of the AAV genome and production of fully infectious virus particles. These AAV-plasmid recombinant DNA molecules should be useful both for site-specific mutagenesis of the viral genome and to study the potential of AAV as a eukaryotic vector.

Adenoviruses, Human↗
PubMed1983

Chromatin-like structure of adeno-associated virus DNA in infected cells.

Adeno-associated virus (AAV) is a single-stranded DNA virus which requires adenovirus as a helper for productive infection. We studied whether intracellular AAV DNA in KB cells was present in a chromatin-like structure by digesting infected cell nuclei with micrococcal nuclease. Virus DNA was detected by agarose gel electrophoresis followed by blotting and hybridization to nick-translated [32P]DNA probes. After coinfection with adenovirus, AAV DNA was present in nucleosome-like structures which were similar to cell nucleosomes and were double stranded as judged by insensitivity to S1 nuclease digestion. In the absence of adenovirus, intracellular AAV DNA also formed similar nucleosome-like structures which were also insensitive to S1 digestion and were formed in both the presence and absence of hydroxyurea. These latter structures probably formed on AAV duplexes created either by reassociation of infecting parental single-stranded DNA or by covalent integration into the cell genome rather than by de novo AAV DNA synthesis. These results have implications for the mechanism of AAV genome replication, transcription, and integration into the cell genome.

Cell Line↗
PubMed1982

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.

Adenoviruses, Human↗
PubMed1981

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA.

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.

Canavanine↗
PubMed1981

Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein.

Growth of adeno-associated virus (AAV) requires expression of certain adenovirus (Ad) genes in the same cell. AAV particles contain three proteins, VP1 (Mr 85,700), VP2 (Mr 72,000), and VP3 (Mr 61,500). These proteins have overlapping peptide maps. We recently reported that AAV RNAs make up a 3'-coterminal family of overlapping molecules. We report here that the most abundant AAV mRNA, a 2.3-kilobase spliced RNA, codes for all three proteins--VP1, VP2, and VP3--when translated in an in vitro reticulocyte lysate. This shows that the AAV capsid proteins are coded by the genome sequence between map positions 48.0 and 96.0 (1 map unit is 1% of the genome or 47 base pairs). When AAV was grown in human KB cells with the Ad temperature-sensitive mutant Ad5ts125 at the nonpermissive temperature (40 degrees C), the accumulation in vivo of AAV capsid proteins VP1, VP2, and VP3 was decreased to less than 1/50th. However, normal amounts of the 2.3-kilobase mRNA were accumulated, and this RNA could be efficiently translated in an in vitro reticulocyte lysate system to yield VP1, VP2, and VP3. These experiments suggest that in infected cells control is exerted upon the AAV 2.3-kilobase mRNA at the translational level and that this control can be influenced by mutations in Ad. These Ad mutations map in the region 2 early gene for the Ad DNA-binding protein. The temperature-sensitive system that we have studied may be useful for analysis of translational control of a eukaryotic mRNA.

Adenoviridae↗
PubMed1981

Adeno-associated virus RNA transcription in vivo.

We have studied RNA transcripts of the defective parvovirus, adeno-associated virus (AAV) present in poly(A)rich and poly-(A)free fractions of nuclear and cytoplasmic RNA prepared from cells infected together with a helper adenovirus. Cytoplasmic poly-(A)rich RNA contains three overlapping spliced AAV RNAs having sizes of 3.9 X 10(3), 3.3 X 10(3) and 2.3 X 10(3) bases respectively. The nuclear precursors of these RNAs appear to be the coterminal unspliced poly(A)-rich RNAs containing 4.2 X 10(3), 3.6 X 10(3) and 2.6 X 10(3) bases respectively. These unspliced RNAs were also found in the cytoplasm. The nuclear poly(A)-free RNA contained a heterogenous population of AAV RNAs that were generally smaller than 2.3 X 10(3) bases. In addition, the Hirt pellet fraction of the nuclear RNA contained two discrete AAV poly(A)-free RNAs having sizes of 2.5 X 10(3) and 2.8 X 10(3) bases. The 4.2 X 10(3) and 3.6 X 10(3)-base unspliced RNAs are more abundant than the coterminal 3.9 X 10(3) and 3.3 X 10(3)-base spliced RNAs whereas the 2.3 X 10(3)-base spliced RNA is much more abundant than the 2.6 X 10(3)-base unspliced RNA. Thus, the cytoplasmic abundance of the AAV spliced RNAs appears to be controlled in part by the post-transcriptional events of splicing or message stability. We also analysed the effects of AAV defective-interfering genomes upon AAV transcription. These studies showed that when synthesis of standard AAV genomes was inhibited more than 10-fold by defective-interfering genomes there was no significant effect on the types or amounts of AAV RNA transcripts which accumulated. These observations indicate that interference by defective-interfering genomes occurs mostly at the level of DNA replication rather than transcription.

Cell Nucleus↗
PubMed1981

Inhibition of adenovirus oncogenicity in hamsters by adeno-associated virus DNA.

Adeno-associated virus (AAV) DNA partially inhibited in Syrian golden hamsters the formation of tumors by the human adenovirus (Ad) type 12. Furthermore, defective interfering AAV particles or their DNA also reduced tumorigenesis. Defective AAV particles contain aberrant genomes with extensive deletions of the internal AAV DNA sequences. Variant AAV DNA, containing 30% of the viral genome, decreased the incidence of Ad-induced tumors from 44 to 18%. Defective AAV particles, which have a buoyant density of 1.32 g/cm3 in CsCl and which are highly enriched for the DNA of the terminal regions (map positions, 0-5 and 95-100), completely suppressed Ad oncogenicity. This observation suggested that the AAV DNA sequences close to the terminal region of the genome mediated the inhibition of the Ad oncogenicity.

Adenoviruses, Human↗
PubMed1980

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.

Adenoviruses, Human↗