The specificity of RNA packaging in isometric RNA plant viruses is principally determined by replication.

A potato virus X (PVX)-based transient expression system (pEff) that produces replicating RNA has been used to examine the specificity of RNA packaging in the isometric viruses, turnip crinkle virus (TCV) and satellite tobacco necrosis virus-1 (STNV-1). Expression of the coat proteins from the subgenomic RNA derived from the replicating PVX genome results in the efficient production of virus-like particles (VLPs), indistinguishable in structure from native virus particles, and encapsidation of both the subgenomic RNA and truncated versions of the replicating genomic RNA. Non-specific encapsidation of host RNA (which is not replicating) could not be detected in this system, implying that replication is the major determinant of packaging in isometric as well as filamentous positive-strand RNA plant viruses. We further utilised the system to investigate the role of putative packaging signals previously identified within the coat protein open reading frames of both TCV and STNV-1. The results show that eliminating the hairpin structures previously identified as packaging signals has no detectable effect on the specificity of RNA packaging. Replacement of the 213 nucleotide sequence within the TCV coat protein coding region, believed to be important for genomic packaging, with an equivalent sequence codon-optimised for Plasmodium falciparum resulted in less efficient capsid formation and RNA packaging, but did not alter packaging specificity; addition of copies of the wild-type sequence did not complement the defects. We propose that replication is the major determinant of genome packaging specificity in plant RNA viruses, while packaging signals may play a role in packaging efficiency.