Using Interferometric Scattering Microscopy to Study the Dynamics of Viruses

Abstract

The structures of many viruses are known in amazing detail, but little is known about the dynamics of how these complex structures assemble and disassemble. This gap in our understanding is due to the difficulties involved in imaging the dynamics of small viruses over the wide range of time scales (subsecond to hours) relevant to their life-cycles. To bridge this gap, we use interferometric scattering microscopy [Lindfors et al. Phys. Rev. Lett. 93, 037401 (2004)] to track, with high temporal resolution and high dynamic range, the positions and sizes of individual viruses. I begin by quantitatively comparing the technique to other optical imaging techniques that can track viruses and nanoparticles. I then discuss the microscope we have built and the additions that enable us to study the dynamics of small viruses. I then describe our experiments on the bacteriophage λ. We show that the interferometric scattering microscope allows us to track the positions of individual λ phages, and that their tracks reflect the structure of the virus. We also demonstrate dynamic measurements of the masses of individual viruses by measuring how λ phages eject their DNA. The final two chapters focus on using interferometric scattering microscopy to understand how viruses assemble. We first examine a model virus system, the P22-Gag particle, developed in Bogdan Dragnea's lab [Saxena et al. Small 12, 5862 (2016)]. With our microscope, we resolve the kinetics of assembly of HIV-1 Gag capsid proteins on the spherical P22 template. The last chapter describes our experiments on the assembly of 28-nm MS2 bacteriophages. We resolve the kinetics of assembly of complete capsids from the coat protein and viral RNA. We find that the MS2 assembly process is strikingly different than that of Gag shells, and we infer some of the mechanisms by which capsid assembly can be disrupted.