Labeling the human respiratory syncytial virus genomic RNA with exogenous probes for fluorescence and electron microscopy
Alonas, Eric James
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A method for labeling the genomic RNA of the human respiratory syncytial virus, as well as for isolating and examining the labeled filamentous virions was achieved. This method utilized the multiply labeled tetravalent probe design, first described in Santangelo et al. 2009. It was shown that by introducing MTRIPs into RSV infected cells immediately before isolating virus, the genomic RNA within individual filamentous virions could be labeled and imaged. This process did not seem to decrease viral titer or affect viral morphology, and allowed for the imaging of the virus using fixed and live cell conventional fluorescence microscopy and super-resolution microscopic techniques such as dSTORM and STED. The imaging of other structural components of the virus, such as the M protein, and as was discovered, the M2-1 protein was also shown. Additionally, the virus was examined for host proteins of the RLR family, which are involved in the cellular innate immune response. It was found that the protein MDA5 was localized in the isolated filaments. Finally, gold nanoclusters were covalently bound to the RNA probe to create a probe that would generate contrast in cryo-TEM and cryo-ET. By hybridizing the probe to an mRNA encoding GFP, complexing it with a cationic lipid transfection agent, and delivering it to cells before plunge-freezing, it was demonstrated that the mRNA-lipoplex granules could be detected. In conclusion, the method allows for both dynamic and ultrastructural information about the viral genome to be gathered.