Electron microscopic characterization of the defectiveness of a temperature-sensitive mutant of Moloney murine leukemia virus restricted in assembly
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Abstract:
The effect of temperature shiftdown on the assembly of ts3 virions was investigated by both scanning (SEM) and transmission (TEM) electron microscopy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studies indicated to be defective in assembly or release of the virions. In the present study, both SEM and TEM revealed the following: (i) there were more cell-associated virions in ts3-infected cells grown at the nonpermissive temperature (39 degrees C) than either in cells grown at the permissive temperature (34 degrees C) or in wild-type MuLV-infected cells grown at 39 degrees C; (ii) there were more normal single particles than multiploids (virions with two or more pieces of genomic RNA) in ts3-infected cells grown at the nonpermissive temperature; (iii) there were more multiploids in ts3-infected cells grown at the nonpermissive temperature than either in cells grown at the permissive temperature or in wild-type MuLV-infected cells grown at the nonpermissive temperature; (iv) upon temperature shift from 39 to 34 degrees C, about 90% of the cell-associated virions dissociated from the cell surface. TEM studies also indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production of multiploids, may not be the reason why ts3 virions accumulate on the cell surface at the nonpermissive temperature. The relatively higher proportion of multiploids found in ts3-infected cells grown at 39 degrees C compared with those grown at 34 degrees C may be due to the higher density of budding virions at the cell surface at the nonpermissive temperature, which increases the possibility of two or more particles assembling close to one another. The accumulation of ts3 virions in all stages of assembly at the nonpermissive temperature, together with the fact that rapid assembly and release of ts3 virions occurred on temperature shiftdown, indicates that virion assembly is restricted after it has been initiated. The probable role of altered glycoprotein(s) in restricting virion assembly is discussed.
Insights
Temperature-sensitive Moloney murine leukemia virus (Mo-MuLV) ts3 virions accumulate at nonpermissive temperatures, but rapid assembly occurs upon temperature shift. This suggests assembly is restricted after initiation, not due to faulty assembly of multiploids.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Moloney murine leukemia virus (Mo-MuLV) is a retrovirus.
- Temperature-sensitive mutants, like ts3, offer insights into viral replication.
- Previous studies suggested ts3 is defective in virion assembly or release.
Purpose of the Study:
- To investigate the effect of temperature shiftdown on ts3 virion assembly.
- To determine the mechanism behind ts3 virion accumulation at nonpermissive temperatures.
Main Methods:
- Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used.
- Cells were infected with ts3 or wild-type Mo-MuLV.
- Experiments involved permissive (34°C) and nonpermissive (39°C) temperatures, including temperature shifts.
Main Results:
- Ts3-infected cells at 39°C showed more cell-associated virions and multiploids compared to permissive temperatures or wild-type.
- Upon shifting from 39°C to 34°C, ~90% of cell-associated virions dissociated.
- TEM revealed rapid virion assembly and release after temperature shiftdown.
Conclusions:
- Faulty assembly leading to multiploids is unlikely the cause of ts3 virion accumulation.
- Multiploid formation may be a consequence of high virion density at the cell surface at 39°C.
- Virion assembly is restricted after initiation, potentially involving altered glycoproteins.