title: Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.
abstract: Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.
authors.affiliation: Division of Biology, Kansas State University, Manhattan 66506, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Gillock
authors.forename: S
authors.initials: S
authors.lastname: Rottinghaus
authors.forename: D
authors.initials: D
authors.lastname: Chang
authors.forename: X
authors.initials: X
authors.lastname: Cai
authors.forename: S A
authors.initials: SA
authors.lastname: Smiley
authors.forename: K
authors.initials: K
authors.lastname: An
authors.forename: R A
authors.initials: RA
authors.lastname: Consigli
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D036022:Capsid Proteins; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D005822:Genetic Vectors; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011120:Polyomavirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C062481:VP1 protein, polyomavirus; D004247:DNA