Original Paper
Intervirology 1992:34:1-12
Cellular and Molecular Biology Research Center (CI BCM), University of Costa Rica, San José, Costa Rica; Department of Virology, Agricultural University, Wageningen, The Netherlands; Department of Plant Pathology, University of Kentucky, Lexington, Ky„ USA; John Innés Institute, Norwich, UK: Department of Applied Biology, University of Hull, Hull, UK; Centre UPC-IRTA, Lleida, Catalunya, Spain
Key Words
CaMV Aphid transmission factor Baculovirus expression
Expression of Cauliflower M osaic Virus ORF II in a Baculovirus System
Summary
The cauliflower mosaic virus O R FII encoding the aphid trans mission factor (ATF) was mutagenized to introduce a BamHl restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda(SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antise rum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (PI8). Subcellular fractionation of cells infected with the recombinant ^cMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified PI 8 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauli flower mosaic virus (CaMV)-infected turnip tissue, this anti serum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing PI 8) and not other plant cellular components.
introduction
Cauliflower mosaic culimovirus (CaMV), the type member of the caulimovirus group [ I], is a DNA-containing plant virus [2]. CaMV is transmitted in a semipersistent manner by sev eral species of aphids [3]. However, purified CaMV is not aphid transmissible, so, as with potyviruses, the presence of an aphid trans mission factor (ATF) is required [4-6]. Various
Received: August 26,1991 Accepted : March 6,1992
experiments have shown that the CaMV ATF is a protein of 18 kD (PI 8) which is encoded by gene II [7-10]. Recently Espinoza et al. [1l]have shown that P I8 is localized in electron-lucent inclusion bodies in infected plant cells. There are many difficulties in purifying PI8 from infected plants, the main ones being the small amounts of the protein and the presence of other host proteins of a similar molecular weight. A baculovirus system based on Auto-
Dr. Ana M. Espinoza Cellular and Molecular Biology Research Center, University of Costa Rica Ciudad Universitaria, San José (Costa Rica)
€>1992 S. Karger AG, Basel 0300 5526/92/ 0341-0001S2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 12:50:24 AM
A.M. Espinozaa, M. Usmany1' T.P. Pi ronec, M. I larve}"* C.J. Woolstonc, V. Medina1 J.M. Vlakh. R. Hull*
Material and Methods Cloning An fcoRI-fiawHI fragment (nucleotides 408-1929 on the sequence of Franck et al. [16]), which contained parts of ORFs 1 and III and all of ORF II of Cabb B-JI isolate, was cloned into M13mpl8. The sequences up stream of CaMV ORF II were mutagenized by the oligonucleotide-directed method of Kunkel [17] to cre ate a BamH I restriction site just next to the 5' end of the translation start codon (ATG; fig 1A). The mutagenesis was confirmed by the dideoxy-DNA sequencing method [18]. The formation of a new BamH\ site allowed cloning of ORF II as a BantHl fragment with out CaMV flanking sequences at the 5' end and 90 nucleotides of CaMV ORF 111at the 3' end (fig. I A).
The baculovirus transfer vector p/itRPKS [12] was used for the recombinant plasmid construction. ORF II was cloned into p/frRPI 8 as a 0.6-kbp BainH\ fragment using the newly created BaniHl site and the natural BamH\ site downstream of ORF II (fig. 1). The replica tive form of M I3m pl8 containing the ORF II was digested with BamYU, and p4
Intervirology 1992:34:1-12
Cellular and Molecular Biology Research Center (CI BCM), University of Costa Rica, San José, Costa Rica; Department of Virology, Agricultural University, Wageningen, The Netherlands; Department of Plant Pathology, University of Kentucky, Lexington, Ky„ USA; John Innés Institute, Norwich, UK: Department of Applied Biology, University of Hull, Hull, UK; Centre UPC-IRTA, Lleida, Catalunya, Spain
Key Words
CaMV Aphid transmission factor Baculovirus expression
Expression of Cauliflower M osaic Virus ORF II in a Baculovirus System
Summary
The cauliflower mosaic virus O R FII encoding the aphid trans mission factor (ATF) was mutagenized to introduce a BamHl restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda(SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antise rum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (PI8). Subcellular fractionation of cells infected with the recombinant ^cMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified PI 8 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauli flower mosaic virus (CaMV)-infected turnip tissue, this anti serum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing PI 8) and not other plant cellular components.
introduction
Cauliflower mosaic culimovirus (CaMV), the type member of the caulimovirus group [ I], is a DNA-containing plant virus [2]. CaMV is transmitted in a semipersistent manner by sev eral species of aphids [3]. However, purified CaMV is not aphid transmissible, so, as with potyviruses, the presence of an aphid trans mission factor (ATF) is required [4-6]. Various
Received: August 26,1991 Accepted : March 6,1992
experiments have shown that the CaMV ATF is a protein of 18 kD (PI 8) which is encoded by gene II [7-10]. Recently Espinoza et al. [1l]have shown that P I8 is localized in electron-lucent inclusion bodies in infected plant cells. There are many difficulties in purifying PI8 from infected plants, the main ones being the small amounts of the protein and the presence of other host proteins of a similar molecular weight. A baculovirus system based on Auto-
Dr. Ana M. Espinoza Cellular and Molecular Biology Research Center, University of Costa Rica Ciudad Universitaria, San José (Costa Rica)
€>1992 S. Karger AG, Basel 0300 5526/92/ 0341-0001S2.75/0
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 12:50:24 AM
A.M. Espinozaa, M. Usmany1' T.P. Pi ronec, M. I larve}"* C.J. Woolstonc, V. Medina1 J.M. Vlakh. R. Hull*
Material and Methods Cloning An fcoRI-fiawHI fragment (nucleotides 408-1929 on the sequence of Franck et al. [16]), which contained parts of ORFs 1 and III and all of ORF II of Cabb B-JI isolate, was cloned into M13mpl8. The sequences up stream of CaMV ORF II were mutagenized by the oligonucleotide-directed method of Kunkel [17] to cre ate a BamH I restriction site just next to the 5' end of the translation start codon (ATG; fig 1A). The mutagenesis was confirmed by the dideoxy-DNA sequencing method [18]. The formation of a new BamH\ site allowed cloning of ORF II as a BantHl fragment with out CaMV flanking sequences at the 5' end and 90 nucleotides of CaMV ORF 111at the 3' end (fig. I A).
The baculovirus transfer vector p/itRPKS [12] was used for the recombinant plasmid construction. ORF II was cloned into p/frRPI 8 as a 0.6-kbp BainH\ fragment using the newly created BaniHl site and the natural BamH\ site downstream of ORF II (fig. 1). The replica tive form of M I3m pl8 containing the ORF II was digested with BamYU, and p4
