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A morphological and immunolabelling study of freeze-substituted human and simian immunodeficiency viruses
C Grief1, M V Nermut, D J Hockley
1Electron Microscopy and Photography Section, National Institute for Biological Standards and Control, Potters Bar, Herts, U.K.
Abstract:
Rapid freezing, freeze substitution and low temperature embedding were used to obtain resin-embedded specimens of HIV and SIV for morphological and immunolabelling studies, with particular emphasis on the 'lateral bodies' and p6 protein. HIV- or SIV-infected cells were fixed in 3% paraformaldehyde and cryoprotected with 0.5 M sucrose. Cells were applied to pieces of Whatman No 1 filter paper and impact-frozen onto a liquid nitrogen cooled copper block. Specimens were freeze-substituted at -90 degrees C using one of three different media: (a) absolute methanol, (b) methanol containing 0.5% uranyl acetate, and (c) methanol containing glutaraldehyde, osmium tetroxide and uranyl acetate. Specimens substituted in methanol and uranyl acetate showed both good structural preservation and retention of antigenicity. We found that the use of filter paper for supporting the specimen was an important factor in obtaining good freezing rates and was more practical than freezing mixtures of cells and gelatin. When compared with specimens prepared by conventional fixation and embedding, freeze-substituted virus particles showed a greater uniformity of shape and size and were more dense in appearance. Distinct 'lateral bodies' were not observed in freeze-substituted viruses. The viral protein p6 was widely distributed in the centre of mature virus particles.
Insights
Rapid freezing and freeze substitution techniques improve the morphological study of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). These methods enhance virus particle preservation and antigenicity for better research insights.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Studying the morphology and protein localization of HIV and SIV is crucial for understanding viral replication and developing antiviral strategies.
- Conventional fixation and embedding methods can introduce artifacts and compromise antigenicity, limiting detailed ultrastructural and immunolabelling studies.
Purpose of the Study:
- To optimize cryofixation and freeze-substitution techniques for high-resolution morphological and immunolabelling studies of HIV and SIV.
- To investigate the ultrastructure of HIV and SIV, focusing on 'lateral bodies' and the distribution of the p6 protein.
Main Methods:
- Rapid freezing (impact-freezing onto liquid nitrogen-cooled copper block) and freeze substitution at -90°C using various methanol-based media.
- Specimen preparation involved cryoprotection with sucrose and support on filter paper for efficient freezing.
- Comparison of freeze-substituted specimens with conventionally fixed and embedded samples.
Main Results:
- Freeze substitution in methanol with uranyl acetate provided excellent structural preservation and retained antigenicity.
- Filter paper support significantly improved freezing rates, offering a practical advantage over gelatin mixtures.
- Freeze-substituted virus particles exhibited enhanced uniformity in shape and size, with increased density compared to conventional methods.
- Distinct 'lateral bodies' were not observed in freeze-substituted viruses.
- The viral p6 protein was found to be distributed throughout the core of mature virus particles.
Conclusions:
- Cryofixation and freeze substitution are superior methods for preserving the fine structure and antigenicity of HIV and SIV.
- The optimized technique allows for more accurate visualization of viral components and protein localization.
- Further research into the role of p6 protein and the absence of 'lateral bodies' in freeze-substituted viruses is warranted.