A study of TMV ts mutant Ni2519. III. Location of the reconstitution initiation sites on Ni2519 RNA.

It was suggested in an accompanying paper [Taliansky et al. (1982b), Virology, 117, 000-000] that two reconstitution initiation sites (RIS I and RIS II) were functionally active in RNA of the temperature-sensitive (ts) TMV mutant Ni2519 upon reassembly at a nonpermissive temperature; initiation of reassembly that started at two sites on the Ni2519 RNA molecule resulted in defective (ribonuclease-sensitive) virus particle (DVP) formation. RIS(s) was(were) defined as a segment(s) of Ni2519 RNA protected from the action of ribonuclease T1 by the coat protein within an incomplete nucleoprotein complex (I-NPC) formed upon limited TMV reassembly at nonpermissive (33 degrees and permissive (24 degrees) temperatures. Tl-resistant oligonucleotides protected within I-NPC were finger-printed, sequenced, and assigned to specific regions on the Ni2519 RNA molecule. It was shown that only RIS I was operative on Ni2519 RNA at 24 degrees as well as on A14 (a temperature-resistant strain, the wild type for Ni2519) RNA at both 24 and 33 degrees ; RIS I corresponded to the so-called Oa (origin of assembly) of a common TMV strain previously studied and was located at a distance of about 800 nucleotides from the 3'-end of the RNA molecule. In contrast, two RISs were revealed on Ni2519 RNA assembled at 33 degrees ; of the two RISs operative in this case the first one is identified as RIS I, while the second (RIS II) is located within 300-520 nucleotides from the 3'-end, i.e., within the coat protein gene. The latter corresponds to the so-called SERF (specifically encapsidated RNA fragment) of the RNA of common TMV.