Distribution of viral RNA molecules during the adenovirus type 5 infectious cycle in HeLa cells

Viral RNA was localized ultrastructurally by in situ hybridization with a biotinylated viral DNA probe and colloidal gold label in HeLa cells during infection with adenovirus type 5. Transcription was monitored by high-resolution autoradiography after short pulses with tritiated uridine. At the earliest stage of virus-induced nuclear transformation, viral RNA was restricted to the small compact fibrillar "early replicative sites" which we had previously demonstrated to be the site of viral DNA replication (Puvion-Dutilleul and Puvion, 1990b). Protease-DNase-treated sections revealed that these fibrillar masses rapidly enlarged and gave rise to a juxtaposed loose fibrillogranular structure devoid of viral RNA. Subsequently, the function of the compact fibrillar zones changed to become the sites of accumulation of single-stranded (ss) viral DNA molecules, and the contiguous new fibrillogranular zones (previously named the peripheral replicative zones, Puvion-Dutilleul and Puvion, 1990a) became not only the centers of replicating viral double-stranded DNA but also the only sites of viral RNA molecules including the elongating RNA transcripts. Pulse-chase experiments revealed an unexpected accumulation of RNA molecules in these extranucleolar regions of infected nuclei.