An in vitro Flaviviridae replicase system capable of authentic RNA replication.
We have established an in vitro replication system for bovine viral diarrhea virus (BVDV), a surrogate for the closely-related hepatitis C virus. In an in vitro reaction, BVDV replication complexes synthesize vRNA and replicative form (RF) and replicative intermediate (RI) RNAs. Kinetic and heparin trapping experiments demonstrate the recycling of RF and RI products and the initiation of vRNA synthesis in this system. Consistent with this, quantitative hybridization reveals the asymmetric synthesis of positive and negative strand RNA products. These findings support the notion that RF serves as a template and RI as a precursor in the synthesis of vRNA. Furthermore, the antiviral activity of an NS5B inhibitor was similar in BVDV replicase and infectivity assays. Together, these results indicate that the in vitro activity of BVDV replicase complexes recapitulates RNA replication that occurs in infected cells, providing a system in which to study both mechanisms and inhibitors of Flaviviridae replication.
Tomassini
J E
JE
Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. joanne_tomassini@merck.com
Boots
E
E
Gan
L
L
Graham
P
P
Munshi
V
V
Wolanski
B
B
Fay
J F
JF
Getty
K
K
LaFemina
R
R
D000818:Animals; D002417:Cattle; D002460:Cell Line; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors; D014779:Virus Replication / Q000187:drug effects
D004791:Enzyme Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D006493:Heparin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase