The Effect of RNA Secondary Structure on the Self-Assembly of Viral Capsids

Previous work has shown that purified capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) is capable of packaging both purified single-stranded RNA molecules of normal composition (comparable numbers of A, U, G, and C nucleobases) and of varying length and sequence, and anionic synthetic polymers such as polystyrene sulfonate. We find that CCMV CP is also capable of packaging polyU RNAs, which-unlike normal-composition RNAs-do not form secondary structures and which act as essentially structureless linear polymers. Following our canonical two-step assembly protocol, polyU RNAs ranging in length from 1000 to 9000 nucleotides (nt) are completely packaged. Surprisingly, negative-stain electron microscopy shows that all lengths of polyU are packaged into 22-nm-diameter particles despite the fact that CCMV CP prefers to form 28-nm-diameter (T = 3) particles when packaging normal-composition RNAs. PolyU RNAs >5000 nt in length are packaged into multiplet capsids, in which a single RNA molecule is shared between two or more 22-nm-diameter capsids, in analogy with the multiplets of 28-nm-diameter particles formed with normal-composition RNAs >5000 nt long. Experiments in which viral RNA competes for viral CP with polyUs of equal length show that polyU, despite its lack of secondary structure, is packaged more efficiently than viral RNA. These findings illustrate that the secondary structure of the RNA molecule-and its absence-plays an essential role in determining capsid structure during the self-assembly of CCMV-like particles.