Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells.
Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells.
复制标题
重组 LuIII 自主细小病毒作为人类细胞的瞬时转导载体。
DOI：
10.1089/hum.1993.4.4-441
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发表时间：
1993
影响因子：
4.2
通讯作者：
Carlson,JO
中科院分区：
文献类型：
--
作者：
Maxwell,IH;Maxwell,F;Rhode3rd,SL;Corsini,J;Carlson,JO
利用与病毒P4启动子相关的荧光素酶或β-半乳糖苷酶报告基因取代传染性克隆（pGLu883）中的病毒编码序列，构建了基于自主细小病毒LuIII基因组的重组体。在用这两种结构中的任何一种与提供LuIII非结构蛋白和衣壳蛋白的质粒共转染的细胞中，发生了重组基因组的切除和复制。转导病毒粒子在共转染细胞的培养基中积累，通过暴露于该培养基的受体细胞的报告细胞活性来测定。LuIII的抗血清可以中和转导活性。在HeLa、293和NB324K细胞共转染后，观察到复制形式DNA和转导病毒粒子的产生，效率依次递增。当重组基因组与辅助构建体中病毒基因两侧的序列存在同源性时，有时会观察到伴随产生具有复制能力的细胞病变病毒。这可以通过去除辅助子的左端同源性来最小化；通过这种方法，制备的荧光素酶转导病毒不受复制能力病毒的影响。使用这些制剂，我们在受体HeLa细胞中观察到荧光素酶的表达（3天后下降）长达7天。重组病毒DNA与链特异性荧光素酶探针的杂交表明两条链都被包装（如LuIII报道的那样），但（-）链有几倍的过剩。我们认为自主转导的细小病毒将在基因转移应用中发挥作用，可能包括只需要瞬时表达的基因治疗。
Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or β-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (–) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.