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A mutated membrane protein of vesicular stomatitis virus has an abnormal distribution within the infected cell and
Abstract:
Two temperature-sensitive (ts) mutants of the M protein of vesicular stomatitis virus (tsG31 and tsG33) are defective in viral assembly, but the exact nature of this defect is not known. When infected cells are switched from nonpermissive (40 degrees C) to permissive (32 degrees C) temperatures in the presence of cycloheximide, tsG33 virus release increased by 100-fold, whereas tsG31 release increased only by 10-fold. Thus, the tsG33 defect is more reversible than that of tsG31. Therefore, we investigated how the altered synthesis and cellular distribution of tsG33 M protein correlates with the viral assembly defect. At 32 degrees C tsG33 M protein is stained diffusely in the cell cytoplasm and later at the budding sites. In contrast, at 40 degrees C the mutant M protein formed unusual aggregates mostly located in the perinuclear regions of virus-infected cells and partially colocalized with G protein in this region. In temperature shift-down experiments, M can be disaggregated and used to some extent for nucleocapsid coiling and budding, which correlates with the virus titer increase. M aggregates also formed after shift-up from 32 to 40 degrees C, indicating a complete dependence of M aggregation on the temperature. Biochemical analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that at 40 degrees C M protein is detected exclusively in pellet fractions (nuclear and cytoskeleton components), whereas at 32 degrees C M protein is mainly in the cytoplasmic soluble fractions. Furthermore, when the temperature is raised from 32 to 40 degrees C, the distribution of M protein tends to shift from the soluble to the pellet and cytoskeletal fractions. Electron micrographs of immunoperoxidase-labeled M protein showed that at 40 degrees C M aggregates are often associated with the outer nuclear membranes as well as with vesicular structures. No nucleocapsid coiling was observed in these cells, whereas coiling and budding were seen at 32 degrees C in cells where M protein was partly associated with the plasma membrane. We suggest that the tsG33 M protein mutation may produce a reversible conformational alteration which causes M protein to aggregate at 40 degrees C, therefore inhibiting the proper association of M protein with nucleocapsids and budding membranes.
Insights
Temperature-sensitive M protein mutants of vesicular stomatitis virus show defects in viral assembly. The tsG33 M protein aggregates at nonpermissive temperatures, hindering viral budding and release.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Vesicular stomatitis virus (VSV) assembly relies on the M protein.
- Temperature-sensitive (ts) mutants tsG31 and tsG33 exhibit assembly defects.
- The precise nature of the M protein defect in these mutants remains unclear.
Purpose of the Study:
- To investigate the correlation between M protein synthesis, cellular distribution, and viral assembly defects in tsG33 VSV.
- To elucidate the mechanism underlying the temperature-dependent aggregation of tsG33 M protein.
Main Methods:
- Temperature shift experiments (nonpermissive to permissive temperatures).
- Immunofluorescence microscopy to visualize M protein localization.
- Biochemical fractionation (pellet vs. soluble fractions) using SDS-PAGE and immunoblotting.
- Electron microscopy of immunoperoxidase-labeled M protein.
Main Results:
- tsG33 M protein forms aggregates at 40°C, primarily in perinuclear regions, and partially colocalizes with G protein.
- At 32°C, tsG33 M protein is diffusely distributed in the cytoplasm and at budding sites.
- M protein shifts from soluble to pellet/cytoskeletal fractions upon temperature increase to 40°C.
- Aggregates associate with nuclear membranes and vesicular structures at 40°C, inhibiting nucleocapsid coiling and budding.
Conclusions:
- The tsG33 M protein mutation induces a reversible conformational change leading to temperature-dependent aggregation.
- M protein aggregation at 40°C disrupts its association with nucleocapsids and budding membranes, causing viral assembly defects.
- The reversibility of tsG33 M protein aggregation correlates with increased viral release upon temperature downshift.