TY - JOUR
T1 - Analysis of classical swine fever virus RNA replication determinants using replicons
AU - Risager, Peter Christian
AU - Fahnøe, Ulrik
AU - Gullberg, Maria
AU - Rasmussen, Thomas Bruun
AU - Belsham, Graham J
PY - 2013/8/1
Y1 - 2013/8/1
N2 - Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.
AB - Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.
KW - Animals
KW - Cell Line
KW - Classical swine fever virus/genetics
KW - Escherichia coli/genetics
KW - Genes, Reporter
KW - Luciferases/analysis
KW - Protein Biosynthesis
KW - RNA, Viral/genetics
KW - Recombination, Genetic
KW - Replicon
KW - Sheep
KW - Staining and Labeling/methods
KW - Transcription, Genetic
KW - Viral Proteins/genetics
KW - Virus Replication
U2 - 10.1099/vir.0.052688-0
DO - 10.1099/vir.0.052688-0
M3 - Journal article
C2 - 23580431
SN - 0022-1317
VL - 94
SP - 1739
EP - 1748
JO - Journal of General Virology
JF - Journal of General Virology
IS - Pt 8
ER -
