Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain

A full-length cDNA clone was constructed from the genome of the human parai nfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombi nant vaccinia virus expressing T7 RNA polymerase, and then the plasmid enco ding 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 wi th fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone wa s demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immed iate downstream of the RNA-editing site of the V gene in the full-length hP IV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cD NA. The rPIV2V(-) possessed a defective V protein that did not have the uni que cysteine-rich domain in its carboxyl terminus (the V-protein-specific d omain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rP1V2V(-) in these cells, however, was partially recovered by adding a nti-interferon (IFN)-beta antibody into the culture medium, showing that th e 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 o f hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) effi ciently replicated in Vero cells, which are known as a IFN-non-producers. H owever, the virus yields of rPIV2V(-) in Vero cells were 10- to 100-fold lo wer than those of control rPIV2, although syntheses of the viral-specific p roteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) vi rions showed anomalous in size as compared with rPIV2 virions. These result s suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis. (C) 2001 Academic Press.