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Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)
Yurii G Kuznetsov1, Shoibal Datta, Natantara H Kothari
1Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
Abstract:
NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.
Insights
Atomic force microscopy revealed how mouse leukemia virus (MuLV) assembles and buds from NIH 3T3 cells. Fixation methods impacted visualization of virions and cell structures during infection.
Area of Science:
- Cell Biology
- Virology
- Microscopy
Background:
- Oncogenic retroviruses like mouse leukemia virus (MuLV) can alter cellular functions.
- Understanding viral morphogenesis and cell interaction is crucial for virology research.
Purpose of the Study:
- To investigate the structural details of MuLV virion assembly and budding from NIH 3T3 cells using atomic force microscopy (AFM).
- To examine the impact of different fixation techniques on visualizing viral structures and cellular components.
Main Methods:
- NIH 3T3 cells were infected with MuLV.
- Cells were fixed using glutaraldehyde alone or postfixed with osmium tetroxide.
- Atomic force microscopy (AFM) was employed for high-resolution imaging.
- Immunolabeling with antibodies and colloidal gold was performed.
Main Results:
- Glutaraldehyde fixation removed the lipid bilayer, revealing virions emerging from the cytoskeleton.
- Osmium tetroxide postfixation preserved the lipid bilayer, allowing visualization of virions attached to the cell surface.
- High-resolution AFM imaging detailed the surface protein assembly of virions.
- Cells infected with a mutated MuLV (gPr80(gag) gene) showed defective virion production and abnormal tubelike structures, indicating failed morphogenesis and budding.
Conclusions:
- AFM is a powerful tool for visualizing retroviral structures and their interaction with host cells.
- Fixation methods significantly influence the structural details observable during viral budding.
- Defective viral gene products can lead to aberrant virion morphogenesis and cell surface abnormalities.
