title: Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.
abstract: 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.
authors.affiliation: Department of Microbiology, Mie University School of Medicine, 2-174 Edobashi, Mie, 514-8507, Japan. kawanom@doc.medic.mie-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Kawano
authors.forename: M
authors.initials: M
authors.lastname: Kaito
authors.forename: Y
authors.initials: Y
authors.lastname: Kozuka
authors.forename: H
authors.initials: H
authors.lastname: Komada
authors.forename: N
authors.initials: N
authors.lastname: Noda
authors.forename: K
authors.initials: K
authors.lastname: Nanba
authors.forename: M
authors.initials: M
authors.lastname: Tsurudome
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: M
authors.initials: M
authors.lastname: Nishio
authors.forename: Y
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D010223:Parainfluenza Virus 2, Human / Q000235:genetics* / Q000302:isolation & purification*; D010957:Plasmids; D017393:RNA Editing; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D018076:DNA, Complementary; C068854:V protein, human parainfluenza virus type 1; D014764:Viral Proteins; D015678:Viral Structural Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine