A morphological and immunolabelling study of freeze-substituted human and simian immunodeficiency viruses

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°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.