Effects of substituting granulin or a granulin-polyhedrin chimera for polyhedrin on virion occlusion and polyhedral morphology in Autographa californica multinucleocapsid nuclear polyhedrosis virus

Abstract

Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 micron) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses.

FIG. 1

AcMNPV transfer plasmids constructed for expression of granulin, granulin-polyhedrin chimera, and polyhedrin genes. (A) pFB/APO, for expression of the AcMNPV polyhedrin gene, under control of the polyhedrin promoter. (B) pFB/TGO, for expression of the TnGV granulin gene, under control of the polyhedrin promoter. (C) pFB/TGO/K, for expression of a modified TnGV granulin gene with a nuclear localization signal. (D) pFB/AT, for expression of a hybrid granulin-polyhedrin gene. pFB/AT contains the 5′ end of the granulin ORF from pFB/TGO and the 3′ end of the polyhedrin ORF from pFB/APO.

FIG. 2

Alignment of predicted sequences for amino acids 21 to 50 of selected granulin and polyhedrin genes. Sequences for the following viruses were obtained from GenBank and aligned with Clustal: AcNPV, AcMNPV; BmNPV, Bombyx mori MNPV; SeNPV, SeMNPV; SfNPV, SfMNPV; MbNPV, Mamestra brassicae MNPV; OpNPV, OpMNPV; HzNPV, Helicoverpa zea SNPV; LdNPV, Lymantria dispar MNPV; ClGV, Cryptophlebia leucotrieta GV; CpGV, Cydia pomonella GV; XcGV, Xestia c-nigrans GV; and PbGV, Pieris brassicae GV. The nuclear localization signal of AcMNPV polyhedrin and homologous regions of other occlusion proteins are boxed. Conserved amino acids are shaded gray.

FIG. 3

Light and electron micrographs of granulin and granulin-polyhedrin chimera crystals produced in Tn5 cells by recombinant AcMNPVs at 4 days postinfection. (A to D) Light micrographs of infected cells with large cuboidal crystals formed by wild-type granulin in the cytoplasm and nucleus (A), crystals formed in the nucleus by granulin containing the nuclear localization signal, KRKK (B), and tetrahedral crystals formed in the nucleus by the granulin-polyhedrin chimera (C and D). (E and F) Electron micrographs showing a large, fractured granulin crystal in the cytoplasm (E) and in the nucleus (F). In panel E, fibrillar structures (FS) are located in the cytoplasm adjacent to the crystal, and numerous unoccluded AcMNPV virions are present just inside the nuclear membrane (arrow). In panel F, fibrillar structures and electron-dense spacers surround a nuclear granulin crystal. (G) AcMNPV virions embedded in a heavily fissured intranuclear granulin crystal. The bars equal 6 μm in panels A to D, 1 μm in panels E and F, and 200 nm in panel G.

FIG. 4

Micrographs of Tn5 cells infected with recombinant AcMNPVs that produce granulin with the KRKK nuclear localization signal (A to C), the granulin-polyhedrin chimera (D to F), or AcMNPV polyhedrin (G to I). (A) Electron micrograph of KRKK-granulin crystals without virions in the nucleus. (B) Association of a fibrillar structure (FS) with electron-dense spacers and a KRKK-granulin crystal. (C) Light micrograph of hemocytes infected with the KRKK-granulin recombinant AcMNPV, adjacent to fat body in a fourth-instar larva of T. ni. Note the large intranuclear KRKK-granulin crystals. (D to F) Electron micrographs of tetrahedral crystals produced by the granulin-polyhedrin chimera. (D) Intranuclear tetrahedral crystals with associated fibrillar structures (FS), electron-dense spacers, and masses of unoccluded virions near the nuclear membrane (arrows). (E and F) Corner of a tetrahedral crystal illustrating dense structure (E), and virions discernible in the central area of a crystal (F). (G to I) Light and electron micrographs illustrating characteristic intranuclear polyhedron formation (G and H) and virion occlusion (H and I) by the AcMNPV recombinant virus that produces AcMNPV polyhedrin. The bars equal 1 μm in panels A and B, 3 μm in panel C, 300 nm in panels D to F, 10 μm in panel G, 1 μm in panel I, and 300 nm in panel H.