Visualizing coronavirus RNA synthesis in time by using click chemistry

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Coronaviruses induce viral RNA replication within cellular structures like double-membrane vesicles (DMVs).
• Nonstructural proteins (nsp's) and double-stranded RNA (dsRNA), a presumed intermediate, localize to these sites.
• The precise location of viral RNA synthesis within these structures remains unclear due to a lack of detected pores.

Purpose of the Study:

• To investigate the sites of coronavirus RNA synthesis within infected cells.
• To determine the correlation between dsRNA localization and active viral RNA synthesis.
• To validate click chemistry for detecting nascent viral RNA.

Main Methods:

• Infected cells were fed a uridine analogue to label nascent RNAs.
• Click chemistry was employed to detect and visualize the labeled nascent viral RNA.
• Localization of nascent RNA, nsp's, dsRNA, and the RNA-dependent RNA polymerase (nsp12) was analyzed.

Main Results:

• Nascent viral RNA and nsp's colocalized with or were adjacent to dsRNA foci early in infection.
• Late in infection, dsRNA became dispersed, and its correlation with nsp's and nascent RNA diminished.
• Foci of nascent RNA consistently colocalized with the RNA-dependent RNA polymerase (nsp12).

Conclusions:

• Click chemistry is a feasible method for detecting viral RNA synthesis in coronavirus-infected cells.
• dsRNA dots do not exclusively represent sites of active viral RNA synthesis.
• Late in infection, DMVs may contain dsRNA that is no longer involved in active RNA synthesis.