Regions of the herpes simplex virus scaffolding protein that are important for intermolecular self-interaction

Abstract

The herpes simplex virus type 1 (HSV-1) scaffolding protein encoded by gene UL26.5 promotes the formation of the icosahedral capsid shell through its association with the major capsid protein VP5 and through intermolecular interactions with itself. Inside the capsid shell, the UL26.5 product together with the maturational protease, a minor protein, form a spherical structure which is broken down and released from the capsid during packaging of the viral genome. Selected residues from four internal regions of the HSV-1 scaffolding protein that have significant conservation of amino acids within the scaffolding proteins of alphaherpesviruses were mutated, and the properties of the proteins were examined. Only the HSV-1 scaffolding protein with mutations in the conserved N-terminal domain showed reduced interaction with the varicella-zoster virus homologue in a cell-based immunofluorescence assay, providing the first evidence that this domain in the HSV-1 protein is likely to be involved in intermolecular self-interaction. Scaffolding protein with mutations in this domain or in either of two other domains failed to assemble into scaffold-like particles but retained the ability to self-interact, although the aggregates were significant smaller than most of the aggregates formed by the wild-type protein. These results suggest that there are multiple domains involved in the intermolecular self-association of the HSV-1 scaffolding protein that can act independently of one another. This conclusion was supported by the observation that none of the mutant proteins with lesions in an individual domain, including a protein with mutations in a central region previously implicated in self-interaction (A. Pelletier, F. Dô, J. J. Brisebois, L. Lagacé, and M. G. Cordingley, J. Virol. 71:5197-5208, 1997), interfered with capsid assembly in a baculovirus expression system. A protein mutated in the central region and another conserved domain, both of which had been predicted to form coiled coils, was impaired for capsid formation but still retained the capacity to interact with VP5.

FIG. 1.

Mutations constructed for analysis of HSV-1 scaffolding protein. At the top, the locations of domains within the HSV-1 scaffolding protein that are conserved in seven alphaherpesviruses and the restriction endonuclease sites used in the construction of missense mutations are shown. The alphaherpesviruses used in the alignment were HSV-1, HSV-2, VZV, equine herpesvirus type 1, equine herpesvirus type 4, pseudorabies virus, and bovine herpesvirus type 1 proteins. Below, the amino acid sequence and location of domains 1 to 4 is shown, with residues that are conserved in all seven viruses displayed on the line below (26). The amino acid changes in the domains used in this study are listed to the right.

FIG. 2.

Colocalization of VZV and HSV-1 scaffolding proteins. Digital confocal images of transfected Vero cells expressing the VZV scaffolding protein (a), the HSV-1 wt scaffolding protein (b), or VZV and HSV-1 proteins (c to h). Bound rabbit polyclonal antibodies (specific for the VZV protein) were visualized with Cy5-GAR IgG (red), and mouse monoclonal antibodies (specific for the HSV-1 protein) were detected with FITC-GAM IgG (green). In both sets of three images for cells dually expressing the VZV and HSV proteins, the image in the right panel represents the merged images in the left and middle panels. Bar, 10 μm.

FIG. 3.

Identification of aggregates of HSV-1 and VZV scaffolding proteins by immunoelectron microscopy. Thin sections of sf21 cells infected with baculoviruses expressing VZV and HSV-1 scaffolding proteins (a and b) and cells singly infected with baculovirus expressing the VZV scaffolding protein (c) or the HSV-1 scaffolding protein (d) were prepared. The samples were incubated with rabbit antiserum specific for VZV scaffolding protein and mouse monoclonal antibody specific for the HSV-1 homologue (a, c, and d) or control antibodies (b) and subsequently treated with GAM IgG conjugated to 10-nm gold particles and GAR IgG conjugated to 30-nm gold particles. Bar, 0.5 μm; large arrow, 30-nm gold particle; small arrow, 10-nm gold particle.

FIG. 4.

Interaction of HSV-1 mutant scaffolding proteins with VZV homologue. Vero cells were transfected with a plasmid expressing a mutant HSV-1 scaffolding protein together with a construct containing the homologous VZV gene, and the proteins were detected using an indirect immunofluorescence assay. Digital confocal images in groups of three represent cells containing the VZV scaffolding protein plus domain 1 triple mutant protein (a, b, c), domain 2 mutant (d, e, f), domain 3 mutant (g, h, i), domain 4 mutant (j, k, l), and domain 2+4 mutant (m, n, o). Bound mouse antibodies (specific for the HSV-1 protein) were visualized with FITC-GAM IgG (green), and bound rabbit antibodies (specific for the VZV protein) were detected with Cy5-GAR IgG (red). In each set, the image in the right panel represents the merged images in the left and middle panels. Bar, 10 μm.

FIG. 5.

Interaction of domain 1 mutants containing single amino acid changes with VZV scaffolding protein. Digital confocal images in groups of three represent transfected Vero cells dually expressing the VZV scaffolding protein and domain 1 mutants Y28A (a, b, c), Q30A (d, e, f), or L31A (g, h, i). Bound mouse monoclonal antibodies (specific for the HSV-1 protein) were visualized with FITC-GAM IgG, and bound rabbit polyclonal antibodies (specific for the VZV protein) were identified using Cy5-GAR IgG. In each set, the image in the right panel represents the merged images in the left and middle panels. Bar, 10 μm.

FIG. 6.

Participation of mutant scaffolding proteins in capsid assembly. Insect cells were infected with a recombinant baculovirus expressing wt or mutant scaffolding protein together with a virus expressing VP5, VP19C, and VP23. Samples were harvested at 50 h postinfection, fixed, and embedded, and thin sections were examined for the presence of capsids under the electron microscope. Shown are electron micrographs of a portion of a cell expressing VP5, VP19C, VP23, and HSV-1 wt scaffolding protein (a), domain 1 mutant protein (b), domain 2 mutant protein (c), domain 3 mutant protein (d), domain 4 mutant protein (e), or domain 2+4 mutant protein (f). Arrows, incomplete capsids; bar, 0.5 μm.

FIG. 7.

Sucrose gradient sedimentation analysis of extracts from insect cells infected with recombinant baculoviruses expressing HSV-1 capsid shell proteins and either wt scaffolding protein (a and c) or mutant 2+4 scaffolding protein (b and d). The extracts were sedimented through 10 to 40% sucrose gradients, and successive fractions were collected. The proteins in each fractions were analyzed by SDS-PAGE and detected by silver staining (a and b), and the scaffolding protein in each fraction was detected by Western blotting using the monoclonal antibody 406 (c and d). The short arrow indicates the major baculovirus capsid protein p39 and the long arrow shows the direction of sedimentation. The positions of the HSV-1 capsid proteins are indicated on the left and in the peak fraction in the wt virus sample. On the right, the positions of the M_r standards are given.

FIG. 8.

Effect of proteolytic cleavage of mutant scaffolds within capsids. Insect cells were multiply infected with a recombinant baculovirus expressing VP5, VP19C, and VP23, a virus expressing the protease, and a virus expressing wt or mutant scaffolding protein. Shown are electron micrographs of a thin section of a cell expressing VP5, VP19C, and VP23, the UL26 gene product, and wt scaffolding protein (a) or domain 1 (b), domain 2 (c), domain 3 (d), or domain 4 (e) mutant scaffolding protein. The arrows point to capsids containing distinctive scaffolds. Bar, 0.5 μm.

FIG. 9.

Aggregates of wt and mutant scaffolding proteins. Insect cells were infected with a single recombinant baculovirus expressing the wt protein or a mutant scaffolding protein and harvested at 72 h postinfection. Extracts were prepared, and the clarified extracts were layered onto 10 to 40% sucrose gradients and centrifuged. Fractions were collected, starting from the top of the gradient. (a) The proteins from each gradient were separated on an SDS-10% polyacrylamide gel, and the scaffolding protein was detected by Western blotting, using monoclonal antibody to the UL26.5 protein. The arrow indicates the direction of sedimentation. (b) The material from selected fractions from each gradient was negatively stained and examined under the electron microscope. Scaffold-like particles were observed in the peak fraction (fraction 7) from gradients of wt protein and domain 3 mutant scaffolding protein only. Bar, 100 nm.

FIG. 10.

Interaction of VP5 with mutant scaffolding proteins. Vero cells were cotransfected with a plasmid expressing VP5 and another expressing the wt protein or a mutant HSV-1 scaffolding protein. Transfections with individual plasmids were also carried out. Proteins were detected using an indirect immunofluorescence assay. The top six digital confocal images represent cells expressing VP5 (a), wt scaffolding protein (b), VP22a (c), domain 1 mutant containing three mutations (d), domain 2+4 mutant (e), and domain 1+2+4 mutant (f). The subsequent confocal images, in series of three, represent cells containing wt scaffolding protein (preVP22a) and VP5 (g, h, i), VP22a and VP5 (j, k, l), domain 1 triple mutant and VP5 (m, n, o), domain 2 + 4 and VP5 (p, q, r), and domain 1+2+4 and VP5 (s, t, u). Bound mouse monoclonal antibodies (specific for the HSV-1 protein) were visualized with FITC-GAM IgG, and bound rabbit polyclonal antibodies (specific for VP5) were identified using Cy5-GAR IgG. In each set of three images of dually expressed HSV proteins, the image in the right panel represents the merged images present in the left and middle panels. Bar, 10 μm.