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Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus
Abstract:
A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.
Insights
A monensin-resistant mouse cell line (MO-5) significantly reduces vesicular stomatitis virus (VSV) replication. This genetic model offers insights into viral endocytosis and transport mechanisms.
Area of Science:
- Cell Biology
- Virology
- Genetics
Background:
- Monensin is an ionophoric antibiotic affecting cellular processes.
- Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) are enveloped viruses.
- Cellular endocytosis and intracellular transport are crucial for viral infection.
Purpose of the Study:
- To investigate the role of monensin resistance in viral replication.
- To characterize the endocytic pathway defect in a monensin-resistant cell line (MO-5).
- To explore the potential of MO-5 cells as a model for studying viral entry and transport.
Main Methods:
- Generation of a monensin-resistant mutant cell line (MO-5) from Balb/3T3 cells.
- Infection of MO-5 and Balb/3T3 cells with VSV and SFV.
- Measurement of viral particle yield and viral RNA synthesis.
- Analysis of intracellular viral localization using Percoll gradient centrifugation.
- Assessment of viral degradation rates.
Main Results:
- MO-5 cells exhibited a 100-fold reduction in VSV yield and RNA synthesis.
- Low pH treatment did not restore VSV production in MO-5 cells.
- VSV accumulated in the endosome/Golgi fraction in MO-5 cells, unlike the lysosome in Balb/3T3 cells.
- Degradation of VSV was significantly impaired in MO-5 cells compared to Balb/3T3 cells.
Conclusions:
- Monensin resistance in MO-5 cells confers resistance to VSV and SFV infection.
- The defect in MO-5 cells affects post-entry steps, likely related to lysosomal trafficking or degradation.
- MO-5 cells provide a valuable genetic tool for dissecting the mechanisms of viral endocytosis and intracellular transport.