Assembly of hybrid RNAs with tobacco mosaic virus coat protein: Evidence for incorporation of disks in 5′-elongation along the major RNA tail

Abstract

We have shown that during the reassembly of tobacco mosaic virus (TMV) RNA, with the coat protein supplied as a “disk preparation”, the lengths of RNA protected from nuclease are “quantized” with steps which correspond to incorporation of the subunits from either a single or, more commonly, both rings of a disk. This interpretation has been challenged and it was suggested that the pattern was due to special, though unspecified features of the sequence of TMV RNA. To test whether the specific sequence of TMV RNA is important during the elongation, rather than just during nucleation, we have now followed growth of particles containing hybrid RNAs, with the TMV RNA origin of assembly but otherwise non-TMV sequences. We have prepared in vitro RNA transcripts containing heterologous RNA 5′ to the origin of assembly sequence from TMV RNA, i.e. with a heterologous RNA tail in place of the natural major 5′-tail and no minor tail, and used these for assembly experiments. In each case we observe a banding pattern very similar to that which we had found with native TMV RNA and with a dominant quantum step of just over 100 bases, and sometimes also a step of 50 bases, strongly suggesting that this is not due to any feature of the TMV RNA. This same repeat is also visible even with a heterologous RNA chosen because it had a sequence repeat of 135 or 136 bases, confirming that the quantization is due to a feature of the elongation reaction and in no way to the RNA sequence being encapsidated. We have also followed elongation with the origin of assembly located 5′ to the heterologous RNA. This leads to a slower elongation along this 3′-tail, after the initial rapid encapsidation of the origin RNA, which lacks any quantization of length protected. These results are fully compatible with the hypothesis we had advanced earlier, that the major growth along the 5′-tail is from preformed aggregates (“disks”) while the minor growth along the 3′-tail is from subunits in the “A-protein” adding singly or a few at a time.