Assembly of tobacco mosaic virus: elongation towards the 3′-hydroxyl terminus of the RNA

Abstract

The assembly of Tobacco Mosaic Virus (TMV) from its isolated coat protein and RNA is initiated at a single site on the RNA [l] located -1000 nucleotides from the 3’-hydroxyl terminus [2]. A consequence of this internal nucleation site is that subsequent elongation must be bidirectional and while the kinetics and mechanism of growth in the major (3’ to 5’) direction have been extensively studied [3], little is known about growth in the reverse (minor) direction except that it occurs at a considerably slower rate [4-71. We have taken advantage of the possibility of isolating fragments of TMV RNA containing the 3’ terminal region to investigate the kinetics of elongation in the 5’ to 3’ direction. Stripping of TMV under mildly alkaline conditions removes protein specifically from the 5’ end of the particle. Thus nuclease-treated Partially Stripped Virus (PSV) contains fragments of the viral RNA with intact 3’-termini but shortened .5’-tails [8]. RNA molecules with very short tails to the 5’ side of the nucleation region show much less rapid elongation than intact molecules [9] but the reaction does still occur [lo]. Taking advantage of this we have examined the kinetics of elongation towards the 3’ terminus using RNA isolated from nuclease-treated PSV rods. The results indicate that assembly in this direction is more rapid when the protein is supplied in the form of small aggregates (A-protein) than when a disk preparation is used.