Measuring the Effects of Viral RNA Structure on Capsid Self-Assembly

Abstract

Positive-sense single-stranded RNA viruses assemble into protein shells that each encapsulate a single molecule of RNA. The assembly of the protein shell, called the capsid, is a complex process, one in which the structure of the viral RNA genome may play a critical role. Owing to the challenging nature of studying RNA folding, capsid assembly, and the effect of one on the other, our understanding of these processes remains limited.

In this PhD thesis, I present a series of new approaches for measuring RNA structure and its effects on virus capsid assembly. In the first half, I describe efforts to probe the complex structures of large RNA molecules using a technique based on the hybridization of short complementary strands of DNA. I use multidimensional DNA microarrays, termed “patch-probe” arrays, to measure couplings between regions of a viral RNA molecule, from which the intramolecular connectivity can be inferred. The resulting map of connections reveals a distribution of short- and long-range connections that are consistent with several previously reported structural models. In the second half of this thesis, I describe investigations of the effects of RNA structure on the yield and kinetics of virus capsid assembly. I show that the kinetics of the nucleation-and-growth assembly pathway, mediated by stem-loop structures within the native RNA structure, can drive selective packaging of the RNA in vitro.

Through these studies, we have developed a richer picture of RNA folding and the roles of RNA structure in virus capsid assembly. Our work has also provided insights into the potential mechanisms by which RNA structure impacts capsid assembly, and has identified avenues for future research in this area.