Visualizing coronavirus RNA synthesis in time by using click chemistry.

Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites. Here, we studied coronavirus RNA synthesis by feeding cells with a uridine analogue, after which nascent RNAs were detected using click chemistry. Early in infection, nascent viral RNA and nsp's colocalized with or occurred adjacent to dsRNA foci. Late in infection, the correlation between dsRNA dots, then found dispersed throughout the cytoplasm, and nsp's and nascent RNAs was less obvious. However, foci of nascent RNAs were always found to colocalize with the nsp12-encoded RNA-dependent RNA polymerase. These results demonstrate the feasibility of detecting viral RNA synthesis by using click chemistry and indicate that dsRNA dots do not necessarily correspond with sites of active viral RNA synthesis. Rather, late in infection many DMVs may harbor dsRNA molecules that are no longer functioning as intermediates in RNA synthesis.