A low resolution model of Rift Valley fever virus solved using electron cryo-microscopy
Introducion and significance. Rift Valley fever virus (RVFV) is a heamorrhagic fever causing virus. It is a zoonotic virus distributed by mosquitoes and infects both humans and animals. RVFV is endemic in sub-Saharan Africa where high mortality rates in farm animals cause a major economic impact. In humans the diseases is usually mild with flu-like symptoms, however, its most severe form (hemorrhagic fever) is often fatal. Reported fatality rates have typically been 1-2% but in some cases as high as 14%. A recent outbreak in Northern Egypt raised concerns that it may spread to Mediterranean countries. Currently there are no vaccines or drugs available for human use.
Experimental approach. In this project we will use a combination of structural and biophysical methods to study the virus structure and cell entry mechanism. High-resolution electron cryo-microscopy structures solved at different stages of virus entry, including apo, receptor-bound, and post-fusion state will shed light on the cell entry process. When combined with fitting of X-ray crystallographic structures of the receptor and the two viral glycoproteins, these structures will provide targets for rational drug design. Electron cryo-tomography will be used to study the membrane fusion process in a virus-liposome model system. To understand the kinetics and lipid composition requirements for fusion, the virus-liposome system will be analysed using a fluorescence based fusion assay.
Bio-safety and site of work. We will use a clone of RVFV that is attenuated and thus safe to work with, but yet functional in fusion assays and structurally identical to the wild type virus. All the work will be carried in the level 3 biosafety containment of Oxford Particle Imaging Centre (OPIC). The centre offers state-of-the-art facilities for electron cryo-microscopy sample preparation, for data acquisition using a FEI Polara 300kV electron microscopy and for fluorescense-based fusion assays using an Agilent spectrofluorometer.
Protein Science & Structural Biology
Project reference number: 306
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Juha T Huiskonen | Structural Biology | Oxford University | UK | juha@strubi.ox.ac.uk |
2011. Rift valley fever: recent insights into pathogenesis and prevention. J. Virol., 85 (13), pp. 6098-105. Read abstract | Read more
Rift Valley fever virus (RVFV) is a zoonotic pathogen that primarily affects ruminants but can also be lethal in humans. A negative-stranded RNA virus of the family Bunyaviridae, this pathogen is transmitted mainly via mosquito vectors. RVFV has shown the ability to inflict significant damage to livestock and is also a threat to public health. While outbreaks have traditionally occurred in sub-Saharan Africa, recent outbreaks in the Middle East have raised awareness of the potential of this virus to spread to Europe, Asia, and the Americas. Although the virus was initially characterized almost 80 years ago, the only vaccine approved for widespread veterinary use is an attenuated strain that has been associated with significant pathogenic side effects. However, increased understanding of the molecular biology of the virus over the last few years has led to recent advances in vaccine design and has enabled the development of more-potent prophylactic measures to combat infection. In this review, we discuss several aspects of RVFV, with particular emphasis on the molecular components of the virus and their respective roles in pathogenesis and an overview of current vaccine candidates. Progress in understanding the epidemiology of Rift Valley fever has also enabled prediction of potential outbreaks well in advance, thus providing another tool to combat the physical and economic impact of this disease. Hide abstract
2009. Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: evidence for GN-GC glycoprotein heterodimers. J. Virol., 83 (8), pp. 3762-9. Read abstract | Read more
Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G(N) and G(C), organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G(N) and G(C) homodimers or G(N)-G(C) heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G(N)-G(C) heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens. Hide abstract