An atomic model of brome mosaic virus using direct electron detection and real-space optimization

Wang, Zhao; Hryc, Corey F.; Bammes, Benjamin; Afonine, Pavel V.; Jakana, Joanita; Chen, Dong Hua; Liu, Xiangan; Baker, Matthew L.; Kao, C. Cheng; Ludtke, Steven J.; Schmid, Michael F.; Adams, Paul D.; Chiu, Wah

doi:10.1038/ncomms5808

Cited by 107 publications

(113 citation statements)

References 48 publications

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“…In the BMV crystal structure, the N-terminal arm under the 5-fold axis in subunit A was largely disordered (37). Similar structural features were also observed in the cryo-EM density maps (38). Therefore, the density observed under the 5-fold axis suggests that the N-terminal arm in CIPtreated B2.3/4 virions gained substantial structural order.…”

Section: Resultssupporting

confidence: 60%

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Hoover

Wang

Middleton

et al. 2016

J Virol

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The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion produc- T he timing of viral genome release into the infected host cell is critically important to the outcome of infection, as it initiates the race between viral processes and the cellular immune responses against the virus. Even small changes in the timing of viral genome release can result in decreased fitness of the virus (1, 2). The regulation of the release of the viral RNAs is poorly understood, especially for icosahedral viruses.Brome mosaic virus (BMV) is a plant-infecting RNA virus that has served as a model system to study the regulation of RNA virus infection (3). A particularly intriguing feature of BMV is that its tripartite positive-strand RNA genome is encapsidated in three distinct virions. All three virions are required for successful infection. Upon entry into cells, RNA1 and RNA2 direct the translation of the viral replication-associated proteins, while RNA3 encodes the movement protein required for cell-to-cell spread and the coat protein (CP). The CP is translated from subgenomic RNA4. In a typical infection, RNA4 is coencapsidated with RNA3 in a 1:1 ratio (4), although the host species can influence the encapsidation of the viral RNAs (5).The BMV CP has multiple regulatory activities during infection (6, 7). Each BMV capsid contains 180 subunits of the CP arranged in a Tϭ3 symmetry. A CP subunit has structural features similar to those of a histone protein. Both have an intrinsically disordered N-terminal arm rich in positively charged residues followed by sequences that fold into a globular domain (7). The N-terminal arm contributes to the differential encapsidation of BMV RNA, has the ability to translocate from the internal cavity to the outside of the virion, and is preferentially cleaved during proteolysis (8,9). Virions formed by CPs that lack the first 8 residues were found to encapsidate RNA2 well but were labile for the encapsidation of RNA1 (8).The BMV virions can be separated by their density into two populations, one enriched for virions containing RNA1, called B1 virions, and the other enriched for virions that encapsidate RNA2 and RNA3/4, called B2.3/4 virions (9). The B1 virions release RNA1 more rapidly than the B2.3/4 v...

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Section: Resultssupporting

confidence: 60%

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Hoover

Wang

Middleton

et al. 2016

J Virol

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“…On the other hand, large particles require more views to sample reciprocal space adequately in three dimensions, according to the πD/d formula of Crowther et al (1970), and these two effects cancel out precisely. Example of a recently-published single-particle cryoEM structure, with images collected using Direct Electron DE-12 direct electron detector -Brome mosaic virus, EMDB-6000 (Wang et al 2014). Densities are shown for a α-helix and two β-strands.…”

Section: Theoretical Backgroundmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

166

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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“…From virions purified in TEN buffer only, 1,385 particles at a pixel size of 2.78 Å/pixel were selected from data imaged on a JEM3200FSC 300-kV cryoelectron microscope (JEOL Ltd., Tokyo, Japan) and a DE-20 camera (Direct Electron, LP, San Diego, CA) at 16 frames per second. Motion correction and damage compensation were performed on a per-particle basis to combine information from the 32 frames per specimen area (22,23). From virions purified in TEN buffer with supplemental magnesium, 2,464 full particles (intact capsid and genome) were manually boxed with EMAN2 (24) from data imaged on a JEM2010F electron cryomicroscope (JEOL Ltd., Tokyo, Japan) and a USA4000 charge-coupled device (CCD; Gatan, Pleasanton, CA) at 3.62 Å/pixel.…”

Section: Methodsmentioning

confidence: 99%

A Newly Isolated Reovirus Has the Simplest Genomic and Structural Organization of Any Reovirus

Auguste

Kaelber

Fokam

et al. 2015

J Virol

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A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T‫1؍‬ FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCEWe describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins. H uman and livestock diseases are commonly caused by viruses transmitted from enzootic hosts by arthropod vectors (1).Mosquito surveillance studies have been key to understanding the prevalence, distribution, and outbreak potential of the majority of these pathogens, as well as for the detection of novel pathogenic and nonpathogenic agents.The family Reoviridae consists of double-stranded RNA (dsRNA) viruses with 9 to 12 genome segments. Reoviruses have both a wide geographic distribution and wide host range, having been isolated from fungi, plants, insects, ticks, arachnids, fish, marine protists, crustaceans, mammals, and birds (2). They are divided into two ...

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An atomic model of brome mosaic virus using direct electron detection and real-space optimization

Cited by 107 publications

References 48 publications

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Single particle electron cryomicroscopy: trends, issues and future perspective

A Newly Isolated Reovirus Has the Simplest Genomic and Structural Organization of Any Reovirus

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