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Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis
Published on: May 24, 2018
Molecular characterization of Drosophila cells persistently infected with Flock House virus
Juan Jovel1, Anette Schneemann
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. jjovel@catie.ac.cr
Abstract:
Little is known about the molecular determinants causing and sustaining viral persistent infections at the cellular level. We found that Drosophila cells persistently infected (PI) with Flock House virus (FHV) invariably harbor defective viral RNAs, which are replicated by the FHV RNA-dependent RNA polymerase. Some defective RNAs encoded a functional B2 protein, the FHV suppressor of RNA interference, which might contribute to maintenance of virus persistence. Viral small interfering RNAs (vsiRNAs) of both polarities were detected in PI cells and primarily mapped to regions of the viral genome that were preserved in the isolated defective RNAs. This indicated that defective RNAs could represent major sources of vsiRNAs. Immunofluorescence analysis revealed that mitochondria and viral proteins are differentially distributed in PI cells and lytically infected cells, which may partly explain the reduction in infectious viral progeny. Our results provide a basis for further investigations of the molecular mechanisms underlying persistent infections.
Insights
Flock House virus (FHV) persistent infections in Drosophila cells involve defective viral RNAs that generate viral small interfering RNAs (vsiRNAs). These defective RNAs and altered protein distribution may explain reduced infectious viral progeny.
Area of Science:
- Molecular virology
- Cell biology
- RNA interference
Background:
- Viral persistent infections are poorly understood at the molecular level.
- Flock House virus (FHV) serves as a model to study viral persistence.
Purpose of the Study:
- To investigate the molecular mechanisms underlying persistent FHV infections in Drosophila cells.
- To identify viral components and cellular changes associated with viral persistence.
Main Methods:
- Analysis of persistently infected (PI) Drosophila cells.
- Detection and sequencing of defective viral RNAs (dvRNAs).
- Quantification of viral small interfering RNAs (vsiRNAs).
- Immunofluorescence microscopy to study protein and organelle distribution.
Main Results:
- PI cells contain dvRNAs replicated by the FHV RNA-dependent RNA polymerase.
- Some dvRNAs encode a functional B2 protein, a suppressor of RNA interference.
- vsiRNAs were detected and mapped to conserved regions within dvRNAs, suggesting dvRNAs as their source.
- Differential distribution of mitochondria and viral proteins was observed in PI cells compared to lytically infected cells.
Conclusions:
- Defective viral RNAs and their products, including vsiRNAs and functional B2 protein, play a role in maintaining FHV persistence.
- Altered cellular compartmentalization of viral components may contribute to reduced infectious viral progeny.
- These findings provide a foundation for understanding the molecular basis of viral persistence.
