title: Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors.
abstract: The baculovirus expression system has been used to produce non-structural proteins encoded by bottom-component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.
authors: Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
authors: H
authors: H
authors: van Bokhoven
authors: J
authors: J
authors: Wellink
authors: M
authors: M
authors: Usmany
authors: J M
authors: JM
authors: Vlak
authors: R
authors: R
authors: Goldbach
authors: A
authors: A
authors: van Kammen
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009029:Mosaic Viruses / Q000235:genetics*; D009036:Moths / Q000382:microbiology*; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins