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The morphology of murine oncornaviruses following different methods of preparation for electron microscopy
Abstract:
The effect of different preparative procedures for electron microscopy on the size and shape of murine oncornaviruses has been studied. With conventional negative staining procedures using neutral sodium phosphotungstate, both murine mammary tumor virus and murine leukemia virus appeared in head-and-tail forms, with a peak head diameter of 122 and 130 nm, respectively. Negative staining with uranyl accetate gave round virions with peak diameters of 148 and 130 nm. Prefixed virus was round with peak diameters of 141 and 130 nm, respectively, in phosphotungstate, and 148 and 117 nm, respectively, in uranyl acetate. With thin sections, the peak diameters were 143 and 123 nm. The preservation of the spherical shape of the virus was obtained by glutaraldehyde fixation dehydration in alcholic solutions of uranyl acetate, and critical point drying. Under these conditions the viruses had peak diameters of 99 and 82 nm, respectively. The size of murine mammary tumor virus has always been found to be larger than murine leukemia virus in all preparations except for negative staining with neutral sodium phosphotungstate. Shadowing of the virion preparations revealed considerable flattening of the particles in all cases except for critical point drying. Negatively stained preparations did not cast any shadow, and thus thethickness of the particles could not be evaluated. Virus can be reversibly converted from spherical to head-and-tail forms by altering osmotic strength. Under most of the conditions used, murine mammary tumor virus gave a bimodal size distribution with significant numbers of particles that were smaller than the major virus size.
Insights
Electron microscopy preparative methods significantly alter murine oncornavirus size and shape. Critical point drying best preserves spherical morphology, yielding smaller particle dimensions for murine mammary tumor virus and murine leukemia virus.
Area of Science:
- Virology
- Electron Microscopy
- Biophysics
Background:
- Murine oncornaviruses, including murine mammary tumor virus (MMTV) and murine leukemia virus (MLV), are important models for studying retroviral structure.
- Accurate determination of viral size and shape is crucial for understanding their morphology and infectivity.
Purpose of the Study:
- To investigate the impact of various electron microscopy preparation techniques on the measured size and shape of MMTV and MLV.
- To identify methods that best preserve the native morphology of these viruses.
Main Methods:
- Comparative analysis of MMTV and MLV morphology using conventional negative staining (neutral sodium phosphotungstate, uranyl acetate), thin sectioning, and fixation followed by dehydration and critical point drying.
- Particle size was determined by measuring peak diameters from electron micrographs.
- Shadowing techniques were employed to assess particle flattening.
Main Results:
- Negative staining resulted in varied shapes (head-and-tail or round) and sizes, influenced by the staining agent.
- Thin sectioning and prefixed samples yielded larger apparent diameters compared to optimized fixation and critical point drying.
- Critical point drying, combined with glutaraldehyde fixation and uranyl acetate dehydration, best preserved spherical morphology, resulting in the smallest measured diameters (99 nm for MMTV, 82 nm for MLV).
- MMTV was generally larger than MLV, except with neutral sodium phosphotungstate staining.
- Shadowing revealed particle flattening, except with critical point drying.
Conclusions:
- Electron microscopy preparation methods critically influence the perceived size and shape of murine oncornaviruses.
- Critical point drying is the preferred method for preserving the native spherical morphology of MMTV and MLV.
- Variations in size and shape observed across different techniques highlight the importance of method selection in ultrastructural studies.