In vitro studies of single-stranded RNA virus self-assembly

Viruses are self-replicating nucleoprotein complexes that have the ability to cause diseases and are composed of, at least, a single copy of genetic material (either RNA or DNA) and a virally encoded proteinaceous (capsid protein [CP]) protective shell. In single-stranded RNA (ss-RNA) viruses genome packaging is unequivocally coupled to capsid assembly. This spontaneous process is driven by CP-CP and CP-RNA interactions. This thesis is concerned with the in vitro self-assembly of a ss-RNA virus, cowpea chlorotic mottle virus (CCMV), and the effects of the length of the nucleic acid on capsid assembly. We show that the size of a capsid depends not only on the preferred curvature of the CP, but also on the length and number of packaged RNA molecules. By carrying out a series of titration curves of CP with RNAs of varying lengths we find that such high encapsidation yield is possible if and only if the assembly reaction is carried out under very specific experimental conditions, namely at a CP:RNA mass-ratio of 6:1 ("Magic ratio"). By doing head-to-head competition experiments, between a reference and a competitor RNA, under limiting assembly conditions we show that the relative packaging efficiency of ssRNA by CCMV CP is a non-monotonic function of RNA length. The relative packaging efficiency increases as the length of the competitor RNAs approaches that of the wild-type RNA (3.2 kilobases [kb]), while for equal-length RNAs it depends on the secondary and tertiary structure of the RNAs. We demonstrate that the length and number of packaged RNAs determines the assembly pathway; unlike the case of long RNAs (> 1.0 kb), when multiple short RNAs (0.5 kb) are packaged by CCMV CP, to virus assembly is a highly cooperative process. This is the first study in which it is shown that in contrast to the accepted dogma the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA complexes but also on the length and number of packaged RNA molecules