Viruses in motion: maturation of an insect virus-like particle – a nanomachine
Virus-like particles (VLPs) are particles that lack the infectious genome of the parent virus but retain the ability to self-assemble. Transient expression of viral coat proteins in a variety of systems can be used to synthesize VLPs that are morphologically indistinguishable from the virus from which they have been derived. In this thesis, I have used VLPs to study virus maturation mechanisms.The main focus of this thesis has been the study of the maturation of Nudaurelia capensis omega virus (NωV), an insect virus. NωV has a pH-controlled maturation mechanism that involves a precise autocatalytic cleavage within the capsid protein that is accompanied by a dramatic decrease in the diameter of the particles. I used insect cell and plant-based transient expression of the NωV coat protein to produce procapsids and showed that reduction in pH results in cleavage of the coat protein and the concomitant decrease in particle diameter. Mature VLPs purified from plants had a structure indistinguishable from the authentic virus at 2.7 Å resolution, as determined by cryo-electron microscopy (cryo-EM). However, the plant-produced VLPs showed the formation of strong interactions between the subunits of NωV, probably representing covalent crosslinks, that limited their dynamic properties. Given the fact that insect cells did not present this difficulty, we used them to make the VLPs for cryo-EM analysis of intermediates in the process of maturation. These analyses revealed the conformational changes in the structure of the particles that accompany maturation, such as the formation of the cleavage site and the closure of the pores of the particle.The culmination of this thesis is represented by a movie of the maturation process of NωV. This movie provides, for the first time, high-resolution information about the large conformational changes and protein rearrangements occurring during the maturation of this virus.