Structural dynamics of viral nanomachines

Abstract

Rotavirus double-layered particles (DLPs) are formed immediately following entry of virions into a host cell. To study the structural dynamics of actively transcribing rotavirus DLPs, we implemented high resolution imaging procedures along with automated computing routines to visualize mRNA synthesis at the nanoscale. Our combined technologies demonstrate a new approach to monitor dynamic structural processes, such as capsid rearrangements, that may be applied to the study of other viruses.

A Non-Symmetric Reconstruction Technique for Transcriptionally-Active Viral Assemblies

“…The raw images (Figure 2A) revealed DLP assemblies having a diameter between 80-90 nm, some of which were associated with RNA strands, consistent with our previous observations [10,13]. A number of particles in the images displayed strongly symmetric features although these attributes varied among the population.…”

“…Complimentary to this result, particles exhibiting externally ordered capsids observed in the same cryo-EM images correlated with very little internally ordered density; and the particles that constituted this structure were surrounded by very little mRNA in the EM images ( Figures 1B and 1C, Class 1). Yet another 3D structure exhibited some external disorder and some internally ordered features, having a modest degree of mRNA in the area surrounding the particles ( Figures 1B and 1C, Class 2) [10]. Here, we build upon these intriguing findings to gain a deeper understanding of protein and genomic RNA arrangements within transcriptionally-active DLPs.…”

“…Recent cryo-EM structural assessments performed by our laboratories revealed the unexpected finding that transcriptionally-active DLPs exhibited varying degrees of disorder in their external VP6 capsids [10]. Additionally, the DLPs with the most disordered external density contained more internally ordered features within the 3D structure.…”

“…Rotavirus (strain SA11-4F) DLPs were prepared as described in our previous work [10]. Transcription activation reactions (25 μl each) were performed in eppendorf tubes and each mixture contained the following components: 1 μg DLPs prepared in 100 mM Tris-HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1 μl RNasin (Promega Corp, Madison, WI).…”

“…EM Affinity grids containing Nickel-nitrilotriacetic acid (Ni-NTA) coatings were prepared using holey carbon grids (C-flat-2/1 grids; Protochips, Inc.) as previously described [10]. The Ni-NTA functionalized layers were comprised of 25% Ni-NTA lipids and 75% 1,2-dilauryl-phosphatidylcholine (DLPC) filler lipids (Avanti Polar Lipids).…”