TY - JOUR

T1 - Modulation of Translation Initiation Efficiency in Classical Swine Fever Virus

AU - Friis, Martin Barfred

AU - Rasmussen, Thomas Bruun

AU - Belsham, Graham J

PY - 2012

Y1 - 2012

N2 - Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild-type (wt) or mutant forms of the IRES of CSFV strain Paderborn were amplified and inserted into dicistronic reporter plasmids encoding Fluc and Rluc under the control of a T7 promoter. The mutations were within domains II, IIId(1), and IIIf of the IRES. The plasmids were transfected into baby hamster kidney (BHK) cells infected with recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase. IRES mutants with different levels of IRES activity were identified and then introduced by homologous recombination into bacterial artificial chromosomes (BACs) containing CSFV Paderborn cDNA downstream of a T7 promoter. From the wt and mutant BACs, full-length CSFV RNA transcripts were produced in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of the wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced changes within the IRES. The growth characteristics of each rescued mutant virus were compared to those of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES have reduced growth in cell culture compared to the wt virus.

AB - Modulation of translation initiation efficiency on classical swine fever virus (CSFV) RNA can be achieved by targeted mutations within the internal ribosome entry site (IRES). In this study, cDNAs corresponding to the wild-type (wt) or mutant forms of the IRES of CSFV strain Paderborn were amplified and inserted into dicistronic reporter plasmids encoding Fluc and Rluc under the control of a T7 promoter. The mutations were within domains II, IIId(1), and IIIf of the IRES. The plasmids were transfected into baby hamster kidney (BHK) cells infected with recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase. IRES mutants with different levels of IRES activity were identified and then introduced by homologous recombination into bacterial artificial chromosomes (BACs) containing CSFV Paderborn cDNA downstream of a T7 promoter. From the wt and mutant BACs, full-length CSFV RNA transcripts were produced in vitro and electroporated into porcine PK15 cells. Rescued mutant viruses were obtained from RNAs that contained mutations within domain IIIf which retained more than 75% of the wt translation efficiency. Sequencing of cDNA generated from these rescued viruses verified the maintenance of the introduced changes within the IRES. The growth characteristics of each rescued mutant virus were compared to those of the wt virus. It was shown that viable mutant viruses with reduced translation initiation efficiency can be designed and generated and that viruses containing mutations within domain IIIf of the IRES have reduced growth in cell culture compared to the wt virus.

KW - Animals

KW - Cell Line

KW - Classical Swine Fever Virus/physiology

KW - Cricetinae

KW - DNA, Complementary/genetics

KW - DNA, Viral/genetics

KW - Gene Expression Regulation, Viral

KW - Genes, Reporter

KW - Genetic Vectors

KW - Mutation

KW - Peptide Chain Initiation, Translational

KW - Plasmids

KW - RNA, Viral/genetics

KW - Swine

U2 - 10.1128/JVI.00346-12

DO - 10.1128/JVI.00346-12

M3 - Journal article

C2 - 22674994

VL - 86

SP - 8681

EP - 8692

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 16

ER -