Coordinated two-disk nucleation, growth and properties, of virus-like particles assembled from tobacco-mosaic-virus capsid protein with poly(A) or oligo(A) of different length

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A)∼ 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high‐molecular‐mass (A)∼ 5000 is used, but becomes a function of chain length particularly with (A)≤185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np= 1/3 (nnt+ 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus‐like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while ‘dislocation’ of the disk to the helical arrangement of capsid subunits (‘helicalization’) and nuclease stability already become established with chains as short as (A)∼ 58 ± 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four‐turn virus‐like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.