Replication of tobacco mosaic virus. VIII. Characterization of a third subgenomic TMV RNA.

In an earlier study we concluded that tobacco mosaic virus (TMV) infections engender a third subgenomic RNA in infected tissue (P. Palukaitis, F. Garcia-Arenal, M. A. Sulzinski, and M. Zaitlin (1983), Virology 131, 533-545). This RNA of approximate MW of 1.1 x 10(6), termed I1-RNA, was shown to be polyribosome-associated and thus was presumed to serve as a messenger RNA in vivo. Upon in vitro translation of I1-RNA in a rabbit reticulocyte lysate system, a major product of MW approximately 50K was generated. When RNA isolated from polyribosomes of infected tissues was analyzed with clones representing distinct regions of the TMV genome, the I1-RNA was shown to be a subset of the TMV genome, representing the 3'-half of the molecule. A TMV-specific DNA fragment (from a phage M13 clone) containing sequences overlapping the 5' end of I1-RNA was used in nuclease S1-mapping experiments with TMV-RNAs isolated from polyribosomes. I1-RNA was thus shown to be a distinct RNA species and not a class of heterogeneous molecules of approximately the same size. The I1-RNA 5' terminus is residue 3405 in the genome. Based on these findings and on consideration of the TMV-RNA sequence, we propose a model for the translation of I1-RNA: after an untranslated sequence of 90 bases, an AUG codon at residues 3495-3497 initiates a protein of MW 54K, terminating at residue 4915. Thus, the amino acid sequence of the 54K protein is coincident with those residues of the carboxy terminus of the well-known 183K TMV protein.