Effect of the Secondary Structure of Long RNAs on their Packaging by Viral Capsid Protein

A large class of viruses use (1000s-of-nucleotides) long single-stranded (ss)RNA molecules as their genomic material, and protect them in self-assembled nucleocapsids. These ssRNAs form large amounts of secondary structure (typically half of the nucleotides are base-paired in duplexes), making them more compact than they would be otherwise. We are interested in the consequences of these secondary structures on the packaging of long ssRNAs by cowpea chlorotic mottle virus (CCMV) capsid protein (CP), which has been shown to package, in vitro, a wide range of lengths and sequences of RNA. In particular, we compare viral-length polyU RNA molecules – which are largely free of secondary structure – with RNAs of normal nucleotide (nt) composition, i.e., comparable numbers of A, U, G, and C. We find that polyU RNAs ranging in size from 1000-10000 nts can be completely packaged by CCMV CP. But in every case they are packaged into virus-like particles (VLPs) with a diameter of about 21nm, despite the fact that the diameter of the VLPs formed from long normal-composition RNA molecules is 28nm. We have also compared the relative packaging efficiencies of polyU and normal-composition RNA the same length, by mixing equal masses of them with an amount of capsid protein sufficient to package either all of one or all of the other, but not all of both. In particular, we compete RNA molecules that are 3000 nts long, the length of the viral genes packaged by this protein. We find that comparable numbers of each molecule are packaged, but the assembly products involve a mix of 21nm and 28nm particles containing polyU RNA and the normal-composition RNA.