Packaging of recombinant RNA molecules into pseudovirus particles directed by the origin-of-assembly sequence from tobacco mosaic virus RNA.

The spontaneous self-assembly mechanism of tobacco mosaic virus (TMV) led us to predict that contiguous foreign RNA sequences of unlimited length would be fully encapsidated into ribonuclease-resistant, virus-like particles ("pseudovirions"), provided the cognate origin-of-assembly sequence from TMV RNA was present. We describe the construction of SP6-transcription plasmids and in vitro synthesis of recombinant RNAs bearing a correctly oriented, 3' terminal, origin-of-assembly from TMV RNA together with 5'-coding regions for chicken egg white prelysozyme, calf preprochymosin or Xenopus borealis rRNA. In the presence of a "disk" preparation of TMV coat protein, complete assembly of the prelysozyme coding sequence into ribonuclease-resistant, rod-shaped pseudovirions was directed specifically by the 3'-TMV RNA sequence. Helical particles of the predicted length were visible in the electron microscope and could be separated by sucrose gradient centrifugation. This in vitro RNA packaging system may offer a more efficient, protective route to introduce foreign mRNAs into whole plants or protoplasts by "pseudoinfection", particularly when studying the transient expression of mRNAs incapable of replicating or spreading from cell to cell. However, we have detected some constraints in the assembly elongation process, especially with highly structured transcripts such as rRNA. The significance of these observations for current views on the cooperative self-assembly mechanism of TMV is discussed.