Correlative light and electron microscopy enables viral replication studies at the ultrastructural level.
Electron microscopy (EM) is a powerful tool to study structural changes within cells caused e.g. by ectopic protein expression, gene silencing or virus infection. Correlative light and electron microscopy (CLEM) has proven to be useful in cases when it is problematic to identify a particular cell among a majority of unaffected cells at the EM level. In this technique the cells of interest are first identified by fluorescence microscopy and then further processed for EM. CLEM has become crucial when studying positive-strand RNA virus replication, as it takes place in nanoscale replication sites on specific cellular membranes. Here we have employed CLEM for Semliki Forest virus (SFV) replication studies both by transfecting viral replication components to cells or by infecting different cell types. For the transfection-based system, we developed an RNA template that can be detected in the cells even in the absence of replication and thus allows exploration of lethal mutations in viral proteins. In infected mammalian and mosquito cells, we were able to find replication-positive cells by using a fluorescently labeled viral protein even in the cases of low infection efficiency. The fluorescent region within these cells was shown to correspond to an area rich in modified membranes. These results show that CLEM is a valuable technique for studying virus replication and membrane modifications at the ultrastructural level.
Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
Kirsi
K
Hellström
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Helena
H
Vihinen
Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.
Katri
K
Kallio
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Eija
E
Jokitalo
Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland. Electronic address: tero.ahola@helsinki.fi.
Tero
T
Ahola
D000330:Aedes / Q000821:virology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D054884:Host-Pathogen Interactions; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D012672:Semliki forest virus / Q000502:physiology / Q000648:ultrastructure*; D014779:Virus Replication
Correlative light and electron microscopy (CLEM); Membrane; Positive-strand RNA viruses; Replication complex