Use of synchrotron radiation to investigate the structure of genomic RNAs inside viral capsids

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

The simplest viruses consist only of nucleic acids surrounded by a protective shell of protein molecules. We are studying the structures, assembly and disassembly of viruses in which the nucleic acid component is a single-stranded (ss) RNA. ssRNA viruses encompass broad classes of infectious agents including those responsible for the common cold, some cancers and AIDS. The most successful line of defence against any virus is vaccination with an appropriate immunogen but in many cases this has proved to be very difficult or impossible. We believe that the improved understanding of the roles of the ssRNA in the assembly and disassembly of such viruses resulting from the work we have recently carried out gives us unique insights that will open up novel therapeutic targets, potentially allowing the efficiency of progeny virus assembly to be reduced dramatically so that non-infectious, partially assembled particles can prime the host's immune system, leading to viral clearance. Central to this effort is an understanding of teh structure(s) of the ssRNA within the viral particle. We are therefore proposing to test whether the main technique available for interogating such structures, X-ray footprinting, can be used on our test viruses. This work involves use of the new Diamond Synchrotron in Oxfordshire where we will build an instrument to allow viruses to be exposed to X-rays in a defined way. The resulting modified and harmless ssRNAs will then be examined in Leeds and by our US collaborators in Oklahoma.

Technical Summary

We are seeking pump-priming support to develop X-ray footprinting of RNA molecules inside the capsids of single-stranded (ss) RNA viruses. As a result of recent pioneering work by the applicants and others, it has become clear that this will be an important technology for the fundamental understanding of RNA viruses. The technique is already widely used in the wider fields of protein-nucleic acid interactions and RNA folding. ssRNA viruses encompass broad classes of infectious agents including those responsible for the common cold, some cancers and AIDS. The most successful line of defence against these viruses is vaccination with an appropriate immunogen but in many cases this has proved to be very difficult or impossible. We believe that the improved understanding of virus assembly and disassembly resulting from the work proposed here will open up novel therapeutic targets, potentially allowing the efficiency of progeny virus assembly to be reduced dramatically so that non-infectious, partially assembled particles can prime the host's immune system, leading to viral clearance.

Planned Impact

Advance in knowledge base We have recently shown that in the capsid of the RNA phage MS2 there is likely to be a well defined structure for the genomic RNA with conserved secondary structural features that are also present in the protein free RNA. This structure appears to play dynamic and critical roles in assisting the phage particle to assembly rapidly and efficiently. Effectively the overlying protein shell and the genomic RNA work as mutual chaperones to make the process highly efficient. This knowledge has important implications for the development of anti-viral drugs directed against viral assembly. There are no such compounds for the ssRNA viruses and one reason is that the field has concentrated efforts on the symmetrical protein components of such systems, neglecting the possible roles that that RNA might play. In order to establish this concept and have it explored more widely it is essential that we a) confirm as far as we can that presence of a defined structure in MS2 and show that this finding is not unique, i.e. that other ssRNA viruses are constructed similarly. In addition it will be important to develop techniques that allow the internal structures of such RNAs to be investigated, eventually in a time-dependent fashion so that changes in response to alterations in physiological conditions can be assessed. Such a technique is available using X-ray synchrotron radiation to create hydroxyl radicals in situ leading to structure/ligand bound sensitive cleavage of the RNA. However, this technique has not yet been reported to work on virus RNAs in capsido. We will develop the technique at the Diamond facility providing the first monochromatic source for such studies and generating an additional resource for UK and other users. It is important to develop this technique within the UK because of the inherent difficulties of transporting live viruses between countries, e.g. to the USA. Benefit to other disciplines X-ray synchrotron footprinting is widely used in the fields of protein-nucleic acid interactions and RNA folding both of which are important in many aspects of fundamental biology. It is therefore extremely likely that other groups will make use of facility and techniques we propose to develop. Dissemination of results Our work will be published in appropriate international journals and will be publicised to other users by staff at Diamond. Social and economic impact Many important scientific advances are only found to be useful many years after the original discovery. The work here focuses on the mechanism of a fundamental biological process and therefore we expect any direct commercial impacts will occur in the longer term. Nevertheless, ssRNA viruses pose significant threats to plant, animal and human health and this work will help to establish novel understanding that can be exploited directly to improve anti-viral therapy.

Publications

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Description The award made us more determined than ever to pursue the use of X-ray footprinting to interrogate the conformations of nucleic acid genomes within viral capsids and during assembly/disassembly. This we have done via access to the dedicated beamline (17-BM) at the Brookhaven Laboratory. No equivalent facility exists in the UK. This has allowed us to determine RNA genome conformations within infectious virions including bacteriophage MS2, TCV and Hepatitis B Virus VLP. The MS2 data have been validated by reference to the asymmetric cryo-EM structure of that virion. We can see the structures seen by cryo-EM, detect additional RNA PS-CP contacts and see that in vitro reassembly does not result in the same RNA conformation as a natural infection, solving a longstanding riddle about the differences between in vivo and in vitro assembly studies.
Exploitation Route We are drafting a methods paper, for Nature Methods, detailing the work that we have done with the MS2 system that hopefully will serve as a source article for other groups to follow.
Sectors Pharmaceuticals and Medical Biotechnology