Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein

Abstract

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

FIG. 1.

Multiplication of the ORFV D1701-V and D1701-Vr10 strains in KOP cells. Infection of KOP cells (multiplicity of infection, 5) with WT D1701-V resulted in increasing plaque formation from 2 (A) to 4 days p.i. (B), whereas 6 days p.i., D1701-Vr10 led only to foci of infected, β-galactosidase-positive cells (C). Cell morphology was recorded under phase-contrast microscopy. The result of mixed infection experiments is shown in panel D. After simultaneous infection of cells with strains D1701-Vr10 and D1701-V in a ratio of 1,000:1, cell lysates were harvested at the indicated times p.i. and titrated. The number of PFU of D1701-Vr10 virus was determined by counting β-galactosidase-positive foci and the number of WT (D1701-V) PFU was directly visualized by counting WT virus plaques.

FIG. 2.

EM of Vero cells infected with D1701-V and D1701-Vr10. Vero cells were infected with strain D1701-V or D1701-Vr10 and thin sections were examined by electron microscopy. Panels A and B illustrate two different regions of cells infected with D1701-V. In panel A, arrows indicate membrane crescents, stars are positioned over IV particles, and an arrowhead indicates enveloped virus within the cell. In panel B, arrows indicate groups of IMVs, and the arrowhead points to an enveloped mature virus. Panels C and D illustrate cell regions infected with D1701-Vr10. Thin arrows point to particles with appended loop-like structures, and the white arrow points to a particle displaying an extension with a visible end. In panel D a star is positioned over a viroplasm partially surrounded by a viral crescent membrane.

FIG. 3.

EM of negatively stained purified ORFV. Panels A to C display ORFV. An envelope surrounds some of the particles and is distinctly visible in panels B and C. Areas of apparent discontinuity in the ball-of-wool structure are indicated (arrowheads). Panel D shows the mutant deleted of the 10-kDa gene with the loop-like structure viewed at different spatial positions; short disorganized tubules are visualized.

FIG. 4.

Cryo-EM of ORFV. Panels A and B show views of untreated WT ORFV. Most particles are enveloped by TGN-derived membranes (arrows). Panels C and E show views of ORFV after prior sonication. The virus particles display a crenellated surface, a faint spiral tubule, and in panel E a few short double filaments on their periphery (arrows). Due to sonication, the envelopes have been removed and most likely recircularized into liposome-like particles. Panel D shows less-dense particles which correspond to empty viral particles. Panel F shows two particles of the virus mutant with their loop-like structures, loosely surrounded by TGN-derived envelopes (arrowheads).

FIG. 5.

Cryo-negative staining EM of ORFV. Panels A to C are views of the WT ORFV. Panels D and E are views of the mutant D1701-Vr10.