Vaccinia virions deficient in transcription enzymes lack a nucleocapsid

Abstract

The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid.

Fig. 1. A model for vaccinia virion structure

(A) The intact MV. No attempt has been made to represent surface tubule elements. (B) The virion core. (C) Cutaway view. The membrane (brown/tan) has been removed from the upper half of the virion, the near end has been removed, and the core wall (yellow/green) has been rendered transparent, thus revealing the multiple layers, the concavities in the core, the lateral bodies (red), and the nucleocapsid (white). (D–F) Sections through the virion in three mutually perpendicular planes. A dynamic 3D model is available on line at www.vacciniamodel.com. From Condit et al. (2006) with permission.

Fig. 2. One step growth characteristics of Cts1

Confluent monolayers of BSC40 cells were infected at moi=10 with either wt or Cts1 and incubated for various times at various temperatures as indicated. Yields were determined by plaque titration at 31°C.

Fig. 3. SDS gel electrophoresis of purified wt and mutant virions

Mutant virus was grown from moi=5 infections under permissive (31°C for Cts1 and +IPTG for vH4i) or non-permissive (37°C for Cts1 and −IPTG for vH4i) conditions and purified by sucrose gradient sedimentation as described in Materials and Methods. 15 ug of virus was loaded in each lane and the gel was stained with Coomassie blue following electrophoresis. MW = molecular weight markers, with molecular weights indicated in kD.

Fig. 4. Assay for DNA in mutant virions

Dilutions of virus were adsorbed to nitrocellulose filters, the filters were processed for hybridization to ³²P labeled vaccinia virus DNA and the hybridization quantified using a phorhporimager. A) Image. B) Quantification.

Fig. 5. Assay of mutant virions for core transcription

Virus was incubated in the presence of NP40 and nucleoside triphosphates containing ³²P CTP, and incorporation of radioactivity into acid insoluble material was measured with respect to time of incubation.

Fig. 6. Immunoblot analysis of wt and mutant virions

Purified wt or mutant virions grown under conditions indicated at the top of the figure were analyzed by immunoblot analysis using antibodies specific to the virion proteins indicated at the left of each row. Immunoblots were developed using chemiluminescence as described in Materials and Methods. The relevant portions of each immunoblot are shown. The assay for H4 did not include wt virions grown at 31°C, and the assay for E1 did not include wt virions grown at 37°C. In the E1 immunoblot the wt 31 and Cts1 31 lanes were originally directly adjacent to each other; these two lanes were separated for the figure to maintain the overall alignment of the columns. The E4L gene produces two proteins comprising the fastest and slowest migrating bands in the immunoblot. The identity of the middle band is unknown.

Fig. 7. Immunoblot analysis of virion protein synthesis in infected cells

Monolayers of BSC40 cells were infected with wt virus or Cts1 at moi=10 and incubated for the various times and temperatures indicated at the top of the figure. Infected cell extracts were analyzed by immunoblot analysis using antibodies specific to the virion proteins indicated at the left of each row. Immunoblots were developed using chemiluminescence as described in Materials and Methods. The relevant portions of each immunoblot are shown.

Fig. 8. Electron microscopic analysis of virion morphology

A) wt infection, 37°C. Bar = 500 nM. B) wt infection, 37°C. Bar = 200 nM. C) vH4i, +IPTG. Bar = 300 nM. D) vH4i, −ITPG. Bar = 200 nM. E) vH4i, −ITPG. Bar = 200 nM. F) Cts1, 37°C. Bar = 300 nM. G) Cts1, 37°C. Bar = 200 nM. Lettering in panels A, C, E–G shows how virions were scored for quantification: u = undefined, n = nucleocapsid, f = full, e = empty, c = collapsed. Panel B highlights two wt virions each with a nucleocapsid, sectioned in mutually perpendicular planes. Panel D contains three collapsed virions from a vH4i infection, −IPTG.