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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Truncation of the nuclear localization signal of polyomavirus VP1 results in a loss of DNA packaging when expressed in the baculovirus system.

Using the pBlueBacIII baculovirus transfer vector, N11-VP1, a truncated form of the polyomavirus major capsid protein VP1, was cloned for expression in the baculovirus-insect cell expression system. The N11-VP1 protein is virtually identical to full-length, wild-type VP1, except that the first 11 amino acids have been deleted from the amino terminus of the protein. The N-terminal region of VP1 has previously been shown to contain the nuclear localization signal (NLS) of the protein and contains residues essential for both nuclear transport as well as DNA-binding functions. The 5-day infected Sf9 cellular lysate from the recombinant N11-VP1 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 as well as Western blotting and was shown to have accurately expressed the N11-VP1 as cloned. Examination of the Coomassie-stained gels revealed that the capsid-like particles composed of the N11-VP1 protein did not contain any host-derived histones. The absence of the histones in the N11-VP1 capsid-like particles is indicative of the inability of these particles to package DNA, a feature which is observed when wild-type VP1 is treated in this manner. Electron microscopy of these particles substantiated this observation. To determine if the deletion of the NLS exhibited true in vivo characteristics, Sf9 insect cells were infected with the recombinant baculovirus carrying the N11-VP1 gene and examined early in infection (30 h post-infection) by indirect immunofluorescence. The N11-VP1 protein was not transported to the nucleus and remained in the cytoplasm. When the Sf9 cells were coinfected with N11-VP1 and polyomavirus VP2 and VP3 carrying baculoviruses, the N11-VP1 was transported to the nucleus by cooperation with the minor capsid proteins. These studies demonstrate that the N-terminal region of VP1, which contains the NLS and DNA-binding domains, is essential for VP1 nuclear transport and its ability to package Sf9 cellular DNA.[1]


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