Bidirectional assembly of tobacco mosaic virus in vitro

THE TIMING OF THE BIDIRECTIONAL GROWTH IN THE ASSEMBLY REACTION OF TOBACCO MOSAIC VIRUS HAS BEEN THE SUBJECT OF CONTROVERSY: Does elongation actually occur simultaneously to 5' and 3' ends or sequentially, first to the 5' end and then to the 3' end? To determine the timing of elongation toward the 3' end directly, using the S1 nuclease mapping method on a cloned cDNA with micrococcal nuclease-digested tobacco mosaic virus RNA, we analyzed encapsidation of the RNA region that was located downstream from the assembly origin. The results clearly showed that elongation toward the 3' end did not occur for at least the first 4 min. Actually it was first observed at 8 min. It is concluded that, in the first 5-7 min, a rapid elongation of the nucleation complex occurs only toward the 5' end of the RNA and that this gives rise to an intermediate particle 260 nm long. Furthermore, the lengths of the RNA that were protected against S1 nuclease digestion showed a clear banding pattern that had a spacing of approximately 100 nucleotides. This supports the hypothesis that the 20S aggregate is kinetically favored as the protein source for elongation to the 3' end.