Visualization of Respiratory Syncytial Virus RNA Synthesis Sites by Ethynyl Uridine Labeling

Modified cell-permeable nucleosides such as 5-bromouridine 5'-triphosphate (BrUTP) or 5-ethynyluridine (5-EU), together with the powerful click chemistry, have been widely used to specifically label newly synthetized RNA in cells and tissues in a simple two-step approach. The use of cellular transcription inhibitors has established metabolic RNA labeling as an optimal approach for the precise visualization of viral RNA transcripts and the microscopic analysis of their distribution in infected cells. The labeling of nascent viral RNA of respiratory syncytial virus (RSV) and other Mononegavirales (MNV) (e.g., rabies virus (RABV), Ebola virus (EBOV), or human metapneumovirus (HMPV)) demonstrated the presence of newly synthesized viral RNAs in virally induced cytoplasmic inclusions called inclusion bodies (IBs). This was the crucial result to confirm that viral RNA synthesis occurs in these IBs, which could thus be renamed viral factories. Here, we describe a method for the unbiased detection of newly synthetized viral RNA in RSV-infected cells via metabolic labeling with 5-EU and subsequent detection using highly specific alkyne-azide "click" chemistry.