Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization

Abstract

The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.

Keywords: Controlled degradation; Immunolocalization of proteins; Vaccinia virus; Virion structure.

Fig. 1

Negative staining of vaccinia virions. Vaccinia virions were adsorbed on a Formvar/carbon coated grid and incubated in the presence of Tris (A–E), NP-40 (F–J) or NP-40 + DTT (K–O). After 20 minutes the grids were washed and stained with PTA, pH 7.0 (A, F, K), PTA, pH 10.5 (B, G, L), AmMo (C, H, M), UA (D, I, N) or NanoW (E, J, O). The grids were examined in a TEM as described in Materials and Methods.

Fig. 2

Electron dense and pore-like structures on the surface of vaccinia core. Vaccinia cores were prepared on grids as described in Fig. 1. A) Vaccinia core stained with UA reveal an electro-dense structure in the middle of the particle. B) A globular structure on the middle of the core was enhanced when the grid was subjected to platinum-carbon shadowing. C) The presence of pore-like structures (arrows) was observed after staining cores with NanoW.

Fig. 3

Analysis by Western blot and immunogold labeling of purified vaccinia virus after controlled degradation of virions. A) Each lane contain the equivalent of 0.06 A_260nm units (~0.7 μg) of virus that was incubated in different buffers as described in Materials and Methods and labeled on the top of the figure. The presence of specific proteins in the pellet (P) or supernatant (S) fractions were determined by Western blot and labeled on each lane. B) Vaccinia virus were adsorbed on grids and incubated with Tris (A, D, G), NP-40 (B, E, H), or NP-40+DTT (C, F, I) as described in Materials and Methods. After this treatment, the proteins associated with the virions were identified by immunogold labeling using the antibodies A27 (●) A4 (•) (A, B, C), A10 (●) F17 (•) (D, E, F), A14 (●) H1 (•) (G, H, I) as described in Materials and Methods.

Fig. 3

Analysis by Western blot and immunogold labeling of purified vaccinia virus after controlled degradation of virions. A) Each lane contain the equivalent of 0.06 A_260nm units (~0.7 μg) of virus that was incubated in different buffers as described in Materials and Methods and labeled on the top of the figure. The presence of specific proteins in the pellet (P) or supernatant (S) fractions were determined by Western blot and labeled on each lane. B) Vaccinia virus were adsorbed on grids and incubated with Tris (A, D, G), NP-40 (B, E, H), or NP-40+DTT (C, F, I) as described in Materials and Methods. After this treatment, the proteins associated with the virions were identified by immunogold labeling using the antibodies A27 (●) A4 (•) (A, B, C), A10 (●) F17 (•) (D, E, F), A14 (●) H1 (•) (G, H, I) as described in Materials and Methods.

Fig. 4

Morphological changes in vaccinia cores after high salt and high DTT treatment. Vaccinia cores prepared as described before were subjected to treatment with 10 mM (A, C) or 100 mM (B, D) DTT in the absence (A, B) or presence (C, D) of 3M NaCl and stained with Nano-W as described in Materials and Methods before visualization by TEM.

Fig. 5

Analysis by Western blot and immunogold labeling of vaccinia cores after treatment with high salt. A) The equivalent of 0.06 A_260nm units (~0.7 μg) of virus that was incubated in NP-40 + DTT to prepare cores as described in Materials and Methods. Cores were then incubated in buffer S (Cntl) or buffer SN (NaCl) as described in Materials and Methods and labeled on the top of the figure. The presence of specific protein in the pellet (P) or supernatant (S) fraction was determined by Western blot. B) Vaccinia virus were adsorbed on grids and incubated with NP-40+DTT to prepare cores as described in Materials and Methods. After this treatment, the grids were incubated in buffer S (A–D) or buffer SN (E–H) for 20 minutes, washed in ddH₂O and proteins associated to the virions were identified by immunogold labeling using the antibodies A10 (●) A4 (•) (A, E), A10 (●) F17 (•) (B, F), A10 (●) H1 (•) (C, G), A10 (●) A3 (•) (D, H) as described in Materials and Methods.

Fig. 5

Analysis by Western blot and immunogold labeling of vaccinia cores after treatment with high salt. A) The equivalent of 0.06 A_260nm units (~0.7 μg) of virus that was incubated in NP-40 + DTT to prepare cores as described in Materials and Methods. Cores were then incubated in buffer S (Cntl) or buffer SN (NaCl) as described in Materials and Methods and labeled on the top of the figure. The presence of specific protein in the pellet (P) or supernatant (S) fraction was determined by Western blot. B) Vaccinia virus were adsorbed on grids and incubated with NP-40+DTT to prepare cores as described in Materials and Methods. After this treatment, the grids were incubated in buffer S (A–D) or buffer SN (E–H) for 20 minutes, washed in ddH₂O and proteins associated to the virions were identified by immunogold labeling using the antibodies A10 (●) A4 (•) (A, E), A10 (●) F17 (•) (B, F), A10 (●) H1 (•) (C, G), A10 (●) A3 (•) (D, H) as described in Materials and Methods.

Fig. 6

Morphological changes on vaccinia cores after protease treatment. Vaccinia cores prepared on the grids as described before were incubated in Tris (A), 2 mg/ml of Pronase E (B) or 2mg/ml Papain (C) for 120 min. After this period, the grids were negative stained with Nano-W as described in Materials and Methods before visualization by TEM.

Fig. 7

Vaccinia DNA is exposed after Pronase E treatment. Vaccinia cores prepared on the grids as described before were incubated in Tris (A, C) or 2 mg/ml of Pronase E (B, D) for 120 min. After this treatment, the grids were washed in ddH₂O and the DNA and proteins associated with the virions were identified by immunogold labeling using the antibodies F17 (●) DNA (•) (A, B), A4 (●) DNA (•) (C, D) as described in Materials and Methods.

Fig. 8

The A3 core protein and vaccinia DNA are exposed after protease treatment. Vaccinia cores prepared on the grids as described before were incubated in Tris (A, D), or 2 mg/ml of Pronase E (B, E), or 2 mg/ml of Papain (C, F) for 120 min. After this period, the grids were washed in ddH₂O and the DNA and proteins associated with the virions were identified by immunogold labeling using the antibodies A3 (●) DNA (•) (A, B, C), L4 (●) DNA (•) (D, E, F) as described in Materials and Methods.

Fig. 9

Temperature sensitive mutant in the core wall protein A3 make fragile particle under non-permissive conditions. Purified Cts8 virions grown at the non-permissive temperature were adsorbed on a Formvar/carbon coated grid and incubated in the presence of Tris (A), or NP-40 + DTT (B–F). After 20 min, the grids were washed, and two were incubated with 40 μg/ml DNase (C, F), for 15 min. The grids were stained with Nano-W (A–C) or UA (D–F) as described in Materials and Methods.

Fig. 10

The core wall protein A3 and DNA are released from the virions after controlled degradation of A3 mutants. Purified wt virions (A–C) and purified Cts8 virions grown at non-permissive temperature were adsorbed on Formvar/carbon coated grids and incubated in the presence NP-40 + DTT. The grids were then washed in ddH₂O and the DNA and proteins associated with the virions were identified by immunogold labeling using the antibodies A10 (●) A4 (•) (A, D), A10 (●) A3 (•) (B, E), A4 (●) DNA (•) (C, F) as described in Materials and Methods.

Fig. 11

L4 protein can be visualized only after DNase treatment. Purified Cts8 virions grown at the non-permissive temperature were adsorbed on Formvar/carbon coated grids and incubated in the presence of NP-40 + DTT. After 20 min, the grids were washed, and not treated (A, B) or treated with 40 μg/ml DNase (C, D). After this period, the grids were washed in ddH₂O and the DNA and proteins associated to the virions were identified by immunogold labeling using the following antibodies L4 (●) DNA (•) (A, C), A3 (●) DNA (•) as described in Materials and Methods.

Fig. 12

A model for vaccinia virus structure. A) Last steps of morphogenesis starting with immature virion (IV), followed by the incorporation of a DNA molecule to form the immature virions with nucleoid (IVN) and the proteolysis of specific proteins to form the mature virion (MV). B) Fine detail of a sagittal section (see inset, lower left) of the MV showing the different virion sub-domains as specified on the left side of the figure. C) Same as B but the virions were treated with NP-40 and the virion lose the lipid content. D) Same as B but the virions were treated with NP-40+DTT and the virus lose the lipids and the membrane proteins.