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Biomedical subjects

R J Samulski

Publications and source records attributed to R J Samulski.

81 records · Page 5Linked to original sources
PubMed1989

Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.

Blotting, Southern↗
PubMed1989

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.

DNA, Recombinant↗
PubMed1988

Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs.

Adenovirus provides helper functions that facilitate replication of adeno-associated virus (AAV). Both the adenovirus E1B 55-Mr and E4 34-Mr polypeptides are required for efficient and timely accumulation of AAV mRNA, proteins, and DNA. The E1B 55-Mr polypeptide is also required for rescue of the integrated AAV genome in Detroit 6-D5 cells in a normal time frame. All of these effects probably result from a single, primary delay in AAV mRNA accumulation. The AAV helper function provided by the E1B 55-Mr and E4 34-Mr polypeptides appears to closely parallel their normal role in the adenovirus replication cycle.

Adenovirus Early Proteins↗
PubMed1987

A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.

Cell Line↗
PubMed1987

Identification of a repeated sequence element required for efficient encapsidation of the adenovirus type 5 chromosome.

Adenovirus type 5 deletion mutants that lack portions of their cis-acting DNA encapsidation signal synthesized nearly normal levels of viral DNA and late polypeptides but failed to efficiently package the DNA into virus particles. A series of mutant viruses carrying small deletions were produced and used to identify a repeated element (AGTAAATTTGGGC and AGTAAGATTTGGCC) as a key component of the packaging signal. One copy of the repeat was sufficient to signal efficient packaging. The packaging domain could function near either end of the viral chromosome but was no longer active when moved several hundred base pairs toward the interior of the DNA molecule.

Adenoviruses, Human↗
PubMed1983

Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV.

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.

Chromosome Deletion↗
PubMed1982

Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells.

We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescue of a latent AAV viral infection. In addition, the efficient rescue of viable AAV from the recombinant plasmid should facilitate the genetic analysis of AAV. Finally, the results of an analysis of the DNA from rescued virions indicate that an inversion of the AAV terminal sequences occurred during replication.

Cells, Cultured↗