Towards in cellulo virus crystallography

Helen M E Duyvesteyn,Armin Wagner,Gwyndaf Evans,Aaron S Brewster,David I Stuart,Geoff Sutton,Juha T Huiskonen,Jonathan M Grimes,Marie-Laure Parsy,Dennis H Bamford,Helen M Ginn,Nicholas K Sauter,Elina Hirvonen,Maija K Pietilä,Johan Hattne

doi:10.1038/s41598-018-21693-3

Abstract

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.

Highlights

Crystals are conventionally grown in vitro, the production of protein crystals in vivo may be achieved via numerous biological processes[7]
Using electron microscopy (EM) and X-ray diffraction, we demonstrate that small crystalline arrays form within cells
Power spectra calculated from 2D projection images of cells confirmed that the condensates possessed crystalline order up to a resolution of ~52 Å (Fig. 2b,c)

Summary

Introduction

Crystals are conventionally grown in vitro, the production of protein crystals in vivo may be achieved via numerous biological processes[7]. The relatively small size of in vivo crystals means that useful diffraction data can be collected only at microfocus synchrotron beamlines or XFELs8–10. For large unit cells and small crystals the intensities of the Bragg peaks will be dramatically reduced (for instance, similar sized crystals of small picornaviruses will have average intensities hundreds of times less than for lysozyme), whereas the background noise increases in proportion to the amount of extraneous material illuminated. We report analysis of cells infected with both wild-type (wt) virus and AmbJ− mutant virus. The latter are unable to package DNA, since they do not possess the packaging protein, J. The experimental setup-up available, whilst unable to fully explore the potential of the method, provided useful information, demonstrating the arrest of PhiX174 maturation at the procapsid state

Methods

Results

Conclusion