Kawano M, Kaito M, Kozuka Y, Komada H, Noda N, Nanba K, Tsurudome M, Ito M, Nishio M, Ito Y
Department of Microbiology, Mie University School of Medicine, 2-174 Edobashi, Mie, 514-8507, Japan.
Virology. 2001 May 25;284(1):99-112. doi: 10.1006/viro.2001.0864.
A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.
从人副流感病毒2型（hPIV2）基因组构建了全长cDNA克隆。首先，用表达T7 RNA聚合酶的重组痘苗病毒感染Vero细胞，然后将编码抗原组序列的质粒与受T7聚合酶启动子控制的聚合酶单位质粒NP、P和L一起转染到Vero细胞中。随后，将转染后的细胞与新鲜的Vero细胞共培养。通过找到引入的遗传标签证明了从cDNA克隆中拯救出重组hPIV2（rPIV2）。作为反向遗传学的应用，我们在全长hPIV2 cDNA中V基因的RNA编辑位点紧邻下游引入了一个核苷酸变化（UCU变为ACU），并能够从该cDNA获得感染性病毒[rPIV2V(-)]。rPIV2V(-)具有缺陷的V蛋白，其羧基末端（V蛋白特异性结构域）没有独特的富含半胱氨酸的结构域。rPIV2V(-)在CV-1和FL细胞中不生长。然而，通过向培养基中添加抗干扰素（IFN）-β抗体，rPIV2V(-)在这些细胞中的复制部分得以恢复，表明rPIV2V(-)对IFN高度敏感，且其在CV-1和FL细胞中不生长主要是由于其对内源性产生的IFN过敏。这些发现表明hPIV2的V蛋白特异性结构域与IFN抗性有关。另一方面，rPIV2V(-)在已知不产生IFN的Vero细胞中高效复制。然而，rPIV2V(-)在Vero细胞中的病毒产量比对照rPIV2低10至100倍，尽管与rPIV2相比，rPIV2V(-)感染的Vero细胞中病毒特异性蛋白及其mRNA的合成在感染后48小时内有所增加。此外，与rPIV2病毒粒子相比，rPIV2V(-)病毒粒子的大小异常。这些结果表明V蛋白在hPIV2的组装、成熟和形态发生中起重要作用。
