Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid-like particles when expressed in the baculovirus system.
The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein-protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein-protein interaction between the expressed proteins. This protein-protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.
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Division of Biology-Ackert Hall, Section of Virology and Oncology, Kansas State University, Manhattan, KS 66506, USA.
Smiley
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Gillock
E T
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Reeves
W M
WM
Consigli
R A
RA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001717:Birds / Q000821:virology; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D018613:Microscopy, Confocal; D011120:Polyomavirus / Q000378:metabolism / Q000502:physiology*; D019065:Virus Assembly / Q000502:physiology*
D036022:Capsid Proteins; C062481:VP1 protein, polyomavirus; C442543:VP2 protein, Polyomavirus