A Simple, RNA-Mediated Allosteric Switch Controls the Pathway to Formation of a T=3 Viral Capsid

Stockley, Peter G.; Rolfsson, Óttar; Thompson, Gary S.; Basnak, Gabriella; Francese, Simona; Stonehouse, Nicola J.; Homans, Steven W.; Ashcroft, Alison E.

doi:10.1016/j.jmb.2007.03.020

Cited by 130 publications

(234 citation statements)

References 47 publications

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“…The in vitro disassembly and reassembly also indicates the involvement of an extrinsic factor other than the ORF2 protein in the assembly of Tϭ3 VLP and the C-C dimer is in a flat conformation that is concomitant with RNA binding. The induction of C-C conformation has been reported with bacteriophage MS2, where the complete assembly of capsid requires the presence of synthetic RNA fragment (39). Therefore, the pentamer of dimer (Fig.…”

Section: Discussionmentioning

confidence: 76%

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway*

Mayazaki

et al. 2010

Journal of Biological Chemistry

151

182

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Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T‫1؍‬ capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T‫3؍‬ icosahedral lattice and contains RNA fragment in contrast to the RNA-free T‫1؍‬ capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids. Hepatitis E virus (HEV),3 the causative agent of acute hepatitis in humans, is primarily transmitted through contaminated water and generally results in epidemic outbreaks in many developing countries. Sporadic cases have also been reported between outbreaks in HEV-endemic regions as well as in nonendemic areas, and these cases are transmitted through zoonotic route. The overall mortality rates of HEV during outbreaks range from 1 to 15% in general, and the highest mortality occurs in pregnant women, with fatality rates of up to 30% (1).HEV consists of a non-enveloped icosahedral capsid and a single-stranded, positive-strand RNA genome of ϳ7.2 kb that encodes three open reading frames (ORFs) (2). The capsid protein, encoded by the ORF2, is composed of 660 amino acids and responsible for most capsid-related functions, such as virion assembly, host interaction, and immunogenicity. Like other hepatitis viruses, HEV is unable to propagate in currently available cell culture systems, and the research of HEV relies largely on the recombinant HEV capsid proteins (3-6). Virus-like particle (VLP) was obtained when the truncated HEV capsid protein was expressed in insect Tn5 cells with deletion of 52 residues from the C terminus and 111 residues from the N terminus (PORF2) (7). Our previous structural analysis of this HEV-VLP by cryo-electron microscopy (cryo-EM) provided a basic understanding of the quaternary arrangement of PORF2, where the reconstructed VLP displayed a Tϭ1 icosahedral particle composed of 60 copies of PORF2 (8). The essential element of PORF2 protein for Tϭ1 VLP assembly includes amino acids 125-600 (9). Recently, the structural information was further refined by the crystal structures of genotype-3 Tϭ1 VLP (10) and genotype-4 Tϭ1 VLP (11), which revealed the tertiary structure of PORF2 to the level of amino acids. However, the Tϭ1 VLPs used in these experiments were much smaller than that of the native virion, which has a diameter of 320 -340 Å, as determined by immuno-EM (12). There is still a need to investigate the assembly pathway of HEV capsid.HEV virion is hypothesized to be made of 180 copies of the capsid protein (8,11). To test this hypothesis, we screened for HEV genoty...

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Section: Discussionmentioning

confidence: 76%

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway*

Mayazaki

et al. 2010

Journal of Biological Chemistry

151

182

View full text Add to dashboard Cite

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“…The structural transitions that occur during maturation and infection are therefore inherently predictable based on an X-ray or cryo-EM structure of the native capsid . These insights are extremely timely, as we are just starting to understand the detailed molecular mechanisms that underlie virus assembly (Basnak et al, 2010;Dykeman & Sankey, 2010;Stockley et al, 2007;Dykeman et al, 2011;Morton et al, 2010;Rolfsson et al, 2010;Toropova et al, 2011) and exploit them. Potential applications include the creation of targeted drug-delivery vehicles and imaging contrast agents (Wu et al, 1995;Lewis et al, 2006), as well as the use of viruses and virus-like particles for vaccine development (Jagu et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…Again, the positions of these points were fixed by scaling to the outside of the protein shell. In MS2, we have shown that the contacts between the genome and the coat proteins in the shell play significant roles in virus assembly (Basnak et al, 2010;Dykeman & Sankey, 2010;Stockley et al, 2007;Dykeman et al, 2011;Morton et al, 2010;Rolfsson et al, 2010;Toropova et al, 2011). We have argued that multiple RNA stem-loop coat protein dimer interactions are required to determine the positions of the A/B quasi-equivalent dimers in the final capsid.…”

Section: Genome Organization Is Also Predictablementioning

confidence: 99%

Structural constraints on the three-dimensional geometry of simple viruses: case studies of a new predictive tool

Keef

Wardman

Ranson

et al. 2013

Acta Crystallogr A Found Crystallogr

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Understanding the fundamental principles of virus architecture is one of the most important challenges in biology and medicine. Crick and Watson were the first to propose that viruses exhibit symmetry in the organization of their protein containers for reasons of genetic economy. Based on this, Caspar and Klug introduced quasi-equivalence theory to predict the relative locations of the coat proteins within these containers and classified virus structure in terms of T-numbers. Here it is shown that quasi-equivalence is part of a wider set of structural constraints on virus structure. These constraints can be formulated using an extension of the underlying symmetry group and this is demonstrated with a number of case studies. This new concept in virus biology provides for the first time predictive information on the structural constraints on coat protein and genome topography, and reveals a previously unrecognized structural interdependence of the shapes and sizes of different viral components. It opens up the possibility of distinguishing the structures of different viruses with the same T-number, suggesting a refined viral structure classification scheme. It can moreover be used as a basis for models of virus function, e.g. to characterize the start and end configurations of a structural transition important for infection.

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“…For the T = 3 MS2, there is a known assembly origin, the 19-nt-long coat protein gene translational operator (TR) stem-loop (10). TR functions as an allosteric effector, switching the dimeric viral capsomere between the two quasi-conformers required to build the capsid (11). This mechanism requires 60 TR-like sites within the genomic RNA for CP contacts (12).…”

mentioning

confidence: 99%

Revealing the density of encoded functions in a viral RNA

Patel

Dykeman

Coutts

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid selfassembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.virus assembly | single-molecule fluorescence correlation spectroscopy | satellite tobacco necrosis virus | packaging signal

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A Simple, RNA-Mediated Allosteric Switch Controls the Pathway to Formation of a T=3 Viral Capsid

Cited by 130 publications

References 47 publications

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway*

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway*

Structural constraints on the three-dimensional geometry of simple viruses: case studies of a new predictive tool

Revealing the density of encoded functions in a viral RNA

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