Establishment and characterization of cytopathogenic and noncytopathogenic pestivirus replicons.
Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease N(pro) in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins- (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding beta-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins- does not induce cell lysis. Accordingly, neither N(pro) nor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins- with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.
Tautz
N
N
Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany. Norbert.Tautz@vetmed.uni-giessen.de
Harada
T
T
Kaiser
A
A
Rinck
G
G
Behrens
S
S
Thiel
H J
HJ
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D018076:DNA, Complementary / Q000235:genetics; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D016679:Genome, Viral; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases; D016133:Polymerase Chain Reaction; D020365:RNA Helicases; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012093:Replicon; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D018076:DNA, Complementary; D005966:Glucuronidase; 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