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.
Srivastava
C H
CH
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Samulski
R J
RJ
Lu
L
L
Larsen
S H
SH
Srivastava
A
A
D015139:Blotting, Southern; D001853:Bone Marrow / Q000382:microbiology; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D006824:Hybridization, Genetic; D015151:Immunoblotting; D007624:KB Cells; D010321:Parvoviridae / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D004279:DNA, Viral