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Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced
Abstract:
Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.
Insights
Temperature-sensitive mouse hepatitis virus A59 (MHV-A59) mutants are less pathogenic. Mutant ts342 shows impaired viral glycoprotein production in astrocytes, leading to reduced replication and virulence.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) exhibit significantly reduced pathogenicity.
- Intracerebral inoculation of ts342 mutant in mice leads to prolonged central nervous system infection, unlike lethal wild-type MHV-A59.
Purpose of the Study:
- To investigate the replication defect of MHV-A59 ts mutant ts342 in primary mouse brain astrocytes.
- To elucidate the molecular mechanisms underlying the attenuation of ts342 mutant in vivo and in vitro.
Main Methods:
- Infection of primary mouse brain astrocytes with ts342 and wild-type MHV-A59.
- Quantification of infectious virus production and cell infection rates.
- Electron microscopy to visualize virion assembly and intracellular structures.
- Analysis of viral RNA and glycoprotein expression.
Main Results:
- ts342 mutant replicated poorly in astrocytes, producing significantly lower infectious virus yields compared to wild-type virus.
- Electron microscopy revealed a lack of mature ts342 virions in infected astrocytes, with no defects in intracellular viral RNA.
- Viral glycoproteins E2 and E1 were undetectable or barely detectable in ts342-infected cells, suggesting a translation or protein stability defect.
- Isolated revertants regained wild-type growth properties in astrocytes and restored lethal pathogenicity in mice.
Conclusions:
- The ts defect in MHV-A59 ts342 mutant is responsible for impaired viral glycoprotein production and replication in astrocytes.
- The ts defect likely affects viral glycoprotein translation or stability, contributing to the mutant's attenuation.
- Replication competence and pathogenicity of MHV-A59 are linked to the proper expression of viral glycoproteins E2 and E1 in host cells.