Srivastava C H, Samulski R J, Lu L, Larsen S H, Srivastava A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):8078-82. doi: 10.1073/pnas.86.20.8078.
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.
为便于对人致病性细小病毒B19进行遗传分析，我们构建了一种杂交B19病毒基因组，其中有缺陷的B19反向末端重复序列被来自非致病性人细小病毒——腺相关病毒2（AAV）的全长反向末端重复序列所取代。杂交AAV - B19基因组从重组质粒中拯救出来，然后在编码AAV DNA复制所需蛋白质的AAV基因（AAV - Rep蛋白）存在的情况下，将DNA转染到腺病毒2感染的人KB细胞中进行复制。此外，在编码病毒衣壳蛋白的AAV基因（AAV - Cap蛋白）存在的情况下，拯救/复制的杂交AAV - B19基因组被包装到成熟的AAV子代病毒粒子中，随后释放到培养上清液中。重组AAV - B19子代病毒粒子对正常人骨髓细胞具有感染性，并在体外强烈抑制红细胞生成。有感染性的重组B19病毒的可得性应有助于对病毒基因组进行突变分析，这反过来可能会产生关于B19诱导发病机制中单个病毒基因功能的信息。杂交AAV - B19基因组也可能被证明是一种用于人骨髓细胞基因转移的有用载体。
