Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VP0, VP3, and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein.
The cDNA sequences encoding the VP0, VP3, and VP1 structural proteins of poliovirus type 3 (strain P3/Leon/37) have been individually cloned and used to prepare recombinant baculoviruses based on Autographa californica nuclear polyhedrosis viruses (AcNPV). Expression of the proteins was monitored in virus-infected Spodoptera frugiperda cells. Both the individually expressed VP0 protein and VP0 derived from the complete poliovirus coding region were shown to incorporate myristic acid. A significant improvement in VP0 protein yield was obtained when the amino terminal glycine of VP0 was changed to alanine (VP0/ala), suggesting that the presence of glycine or its myristylation is unfavorable for VP0 expression. Even so, the expression levels of the poliovirus capsid proteins were low by comparison with those obtained for other foreign genes (e.g., lacZ). The reason does not appear to be due to protein instability or, from the studies undertaken with VP0, the sequences that flank the AUG codon. Using recombinant baculoviruses that express VP0, VP1, and VP3 (or VP0/ala, VP1, and VP3), poliovirus-like particles (VLPs) were isolated from infected S. frugiperda cells. Examination by electron microscopy of the VLPs purified by CsCl gradient centrifugation revealed structures corresponding in size, appearance, and antigenicity to those expected for poliovirus procapsids; however, the yields of particles were low when compared to those derived from a construct (AcLeon) that expresses the complete coding region of poliovirus type 3, indicating that procapsid synthesis from the P1 precursor is a more favored route.
NERC Institute of Virology and Environmental Microbiology, Oxford, United Kingdom.
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S
Bräutigam
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Snezhkov
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DH
Bishop
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D036022:Capsid Proteins; D002460:Cell Line; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection
D036022:Capsid Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; C442539:VP0 protein, Poliovirus; C442540:VP1 protein, Poliovirus; C442542:VP3 protein, Poliovirus