Tobacco (Nicotiana edwardsonii and N. benthamiana) protoplasts infected with the plant rhabdovirus sonchus yellow net virus (SYNV) were found to be suitable for studies of replication. SYNV messenger RNAs could be detected within 2 hr postinoculation (PI), accumulated to a maximum within 24 hr, and subsequently declined to undetectable levels by 60 hr. Plus- and minus-sense genomic RNAs appeared later and were most abundant by 36 hr PI, but the levels decreased by 60 hr. The four major SYNV structural proteins could be detected by Western blot serological analyses by 24 hr PI and were present in highest concentrations between 43 and 60 hr PI. Among various glycosylation inhibitors, only tunicamycin treatment of protoplasts altered viral protein patterns and resulted in synthesis of a glycoprotein with an apparent molecular weight (Mr) 10% smaller than that found in untreated protoplasts. Two specific cleavage products of the nucleocapsid protein estimated to be 21,000 and 37,000 Mr appeared in N. benthamiana-infected protoplasts by 60 hr PI, but in the presence of tunicamycin, the cleavage products were evident by 38 hr. This result suggests that specific cleavage of the N protein is correlated with stress of infected cells due to viral accumulation and/or tunicamycin treatment.