Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.
Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.
Cai
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Section of Virology and Oncology, Kansas State University, Manhattan 66506.
Chang
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Rottinghaus
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Consigli
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RA
D000818:Animals; D002213:Capsid / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D036022:Capsid Proteins; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D011120:Polyomavirus / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D036022:Capsid Proteins; D011994:Recombinant Proteins; C442543:VP2 protein, Polyomavirus; D004247:DNA