Replication-coupled packaging mechanism in positive-strand RNA viruses:: Synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration

viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Synchronized coexpression of wild-type BMV and FHV genome components in plant cells resulted in the assembly of virions packaging the respective viral progeny RNA. To further elucidate the link between replication and packaging, coat protein (CP) open reading frames were precisely exchanged between BMV RNA 3 (B3) and FHV RNA 2 (F2), creating chimeric RNAs expressing heterologous CP genes (B3/FCP and F2/BCP). Coinfiltration of each chimera with its corresponding genome counterpart to provide viral replicase (B1+B2+B3/FCP and F1+F2/ BCP) resulted in the expected progeny profiles, but virions exhibited a nonspecific packaging phenotype. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.