Requirements for nuclear localization and supramolecular assembly of a baculovirus polyhedrin protein.
This study defines the requirements for the nuclear localization, stable nuclear association, and supramolecular assembly of a baculovirus polyhedrin protein in lepidopteran insect cells. Fragments of the polyhedrin protein were genetically fused to two different nonnuclear reporter proteins and the intracellular distribution of the fusion proteins was analyzed in infected insect cells. Analysis by indirect immunofluorescence showed that the domain between amino acids 30 and 57 could mediate nuclear localization of polyhedrin. However, biochemical fractionation experiments showed that this domain was not sufficient for a detergent-stable association of polyhedrin with the nucleus. This required a slightly larger domain, between amino acids 30 and 110. Differential interference-contrast microscopy showed that the supramolecular assembly of polyhedrin into nuclear occlusion-like particles required the domain between amino acids 19 and 110. The most likely candidate for a minimal nuclear localization signal was the sequence KRKK, located between amino acids 32 and 35. Therefore, oligonucleotide-directed mutagenesis was used to change this sequence to NGNN and the intracellular distribution of the mutant protein was analyzed. The results showed that the mutant protein was predominantly localized in the cytoplasm of infected cells, where it assembled into large, cubic, occlusion-like particles. Thus, the KRKK sequence is necessary for the nuclear localization of polyhedrin, but nuclear localization is not required for its supramolecular assembly into occlusion-like particles.
Jarvis
D L
DL
Department of Entomology, Texas A&M University, College Station 77843.
Bohlmeyer
D A
DA
Garcia
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000382:microbiology; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000077183:Occlusion Body Matrix Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D001616:beta-Galactosidase; D005966:Glucuronidase