A study of TMV ts mutant Ni2519. II. Temperature-sensitive behavior of Ni2519 RNA upon reassembly.

The virion RNA of the temperature-sensitive (ts) TMV mutant Ni2519 was used in experiments on in vitro reconstitution. At 24 degrees RNA of Ni2519 can be reassembled into an infective virus. At 33 degrees the reassembly of Ni2519 RNA results in defective (ribonuclease sensitive) virus particle (DVP) formation. The site of the Ni2519 RNA molecule available for RNase attack is located at a distance of about 600-800 nucleotide residues from the 3'-end in DVP assembled at 33 degrees . It has been shown that it is the initiation (rather than elongation) phase of the virus reconstitution that is temperature-sensitive. The results of an electron microscopic analysis suggest that at least two reconstitution initiation sites (RIS) are operative upon Ni2519 RNA assembly at a nonpermissive temperature (33 degrees). On the contrary, a single RIS is observed in Ni2519 RNA at 24 degrees and in TMV vulgare RNA at 33 degrees. It is suggested that the process of reassembly starts from two initiation sites at 33 degrees and results in the defective particle formation. A model of TMV Ni2519 DVP reassembly at restrictive temperature is offered.