Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity.
Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10(5) PFU/microg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.
Mendez
E
E
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Ruggli
N
N
Collett
M S
MS
Rice
C M
CM
D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000821:virology*; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000472:pathogenicity; D010447:Peptide Hydrolases; D010957:Plasmids / Q000235:genetics; D020365:RNA Helicases; D012367:RNA, Viral / Q000235:genetics*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics*; D014774:Virulence / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018076:DNA, Complementary; D010447:Peptide Hydrolases; D020365:RNA Helicases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C086730:p80 protein, bovine viral diarrhea virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases