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Biogenesis of the Semliki Forest virus RNA replication complex
P Kujala1, A Ikäheimonen, N Ehsani
1Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki, Finland.
Journal of Virology
|March 27, 2001
Summary
Semliki Forest virus RNA polymerase components localize to cytoplasmic vacuoles and cell surfaces, suggesting spherules are RNA replication complexes. These complexes may assemble at the plasma membrane and circulate via endosomal pathways.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Semliki Forest virus (SFV) nonstructural (ns) proteins are essential for viral RNA replication.
- Alphavirus RNA polymerase components' precise localization and assembly remain incompletely understood.
Purpose of the Study:
- To determine the intracellular localization of SFV nsP1 to nsP4 proteins.
- To investigate the role of cytoplasmic vacuoles (CPV) and spherules in viral RNA replication.
Main Methods:
- Confocal and immuno-electron microscopy (EM) for ns protein localization.
- Double labeling with organelle-specific markers.
- Immunoprecipitation assays to assess ns protein interactions.
Main Results:
- All ns proteins colocalized with cytoplasmic vacuoles (CPV) and their associated spherules.
- ns proteins and viral RNA were found on the limiting membranes of CPV and within spherules.
- CPV were identified as derivatives of late endosomes and lysosomes.
- Spherules were observed on cell surfaces and released into the medium.
- ns proteins formed a complex, with each precipitating the others.
Conclusions:
- Spherules represent template-associated RNA polymerase complexes.
- Viral RNA replication complexes likely assemble at the plasma membrane and are trafficked via endosomal pathways.
- CPV and spherules play a dynamic role in viral RNA replication and dissemination.
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