scholarly journals The RNA-Binding Protein of a Double-Stranded RNA Virus Acts like a Scaffold Protein

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Carlos P. Mata ◽  
Johann Mertens ◽  
Juan Fontana ◽  
Daniel Luque ◽  
Carolina Allende-Ballestero ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Rna Virus ◽  
Scaffold Protein ◽  
Double Stranded Rna ◽  
Force Microscopy ◽  
Ribonucleoprotein Complexes ◽  
Atomic Force ◽  
Bursal Disease ◽  
Dsdna Viruses ◽  
Icosahedral Capsid

ABSTRACTInfectious bursal disease virus (IBDV), a nonenveloped, double-stranded RNA (dsRNA) virus with a T=13 icosahedral capsid, has a virion assembly strategy that initiates with a precursor particle based on an internal scaffold shell similar to that of tailed double-stranded DNA (dsDNA) viruses. In IBDV-infected cells, the assembly pathway results mainly in mature virions that package four dsRNA segments, although minor viral populations ranging from zero to three dsRNA segments also form. We used cryo-electron microscopy (cryo-EM), cryo-electron tomography, and atomic force microscopy to characterize these IBDV populations. The VP3 protein was found to act as a scaffold protein by building an irregular, ∼40-Å-thick internal shell without icosahedral symmetry, which facilitates formation of a precursor particle, the procapsid. Analysis of IBDV procapsid mechanical properties indicated a VP3 layer beneath the icosahedral shell, which increased the effective capsid thickness. Whereas scaffolding proteins are discharged in tailed dsDNA viruses, VP3 is a multifunctional protein. In mature virions, VP3 is bound to the dsRNA genome, which is organized as ribonucleoprotein complexes. IBDV is an amalgam of dsRNA viral ancestors and traits from dsDNA and single-stranded RNA (ssRNA) viruses.IMPORTANCEStructural analyses highlight the constraint of virus evolution to a limited number of capsid protein folds and assembly strategies that result in a functional virion. We report the cryo-EM and cryo-electron tomography structures and the results of atomic force microscopy studies of the infectious bursal disease virus (IBDV), a double-stranded RNA virus with an icosahedral capsid. We found evidence of a new inner shell that might act as an internal scaffold during IBDV assembly. The use of an internal scaffold is reminiscent of tailed dsDNA viruses, which constitute the most successful self-replicating system on Earth. The IBDV scaffold protein is multifunctional and, after capsid maturation, is genome bound to form ribonucleoprotein complexes. IBDV encompasses numerous functional and structural characteristics of RNA and DNA viruses; we suggest that IBDV is a modern descendant of ancestral viruses and comprises different features of current viral lineages.

scholarly journals Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

2009 ◽  
Vol 386 (3) ◽  
pp. 891-901 ◽  
Author(s):  
Daniel Luque ◽  
Irene Saugar ◽  
María Teresa Rejas ◽  
José L. Carrascosa ◽  
José F. Rodríguez ◽  
...  
Keyword(s):  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Rna Virus ◽  
Infectious Bursal Disease ◽  
Double Stranded Rna ◽  
Ribonucleoprotein Complexes ◽  
Bursal Disease

scholarly journals Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Álvaro Ortega-Esteban ◽  
Carlos P. Mata ◽  
María J. Rodríguez-Espinosa ◽  
Daniel Luque ◽  
Nerea Irigoyen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Nucleic Acids ◽  
Capsid Protein ◽  
Cycle Phase ◽  
Human Virus ◽  
Double Stranded Rna ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy

ABSTRACT Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo. IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

scholarly journals Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase

2021 ◽  
Author(s):  
Joseph G Beton ◽  
Jim Monistrol ◽  
Anne Wentink ◽  
Erin C Johnston ◽  
Anthony J Roberts ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Fibre Surface ◽  
Nucleotide Exchange ◽  
Force Microscopy ◽  
Atomic Force ◽  
Nucleotide Exchange Factor ◽  
Cryo Electron Tomography ◽  
Active Zones ◽  
Human Hsp70

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Recruitment and clustering of Hsc70 on the fibrils appear to be necessary for disassembly. Here we use atomic force microscopy (AFM) to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography reveals fibrils with regions of densely bound chaperones extending from the fibre surface, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~4 subunits per second.

scholarly journals High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6

2018 ◽  
Vol 13 (8) ◽  
pp. 696-701 ◽  
Author(s):  
Adrian P. Nievergelt ◽  
Niccolò Banterle ◽  
Santiago H. Andany ◽  
Pierre Gönczy ◽  
Georg E. Fantner
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Scaffold Protein ◽  
Force Microscopy ◽  
Atomic Force

scholarly journals Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masahiro Nakano ◽  
Yukihiko Sugita ◽  
Noriyuki Kodera ◽  
Sho Miyamoto ◽  
Yukiko Muramoto ◽  
...  
Keyword(s):  
Influenza A ◽  
High Speed ◽  
Rna Synthesis ◽  
Ultrastructural Feature ◽  
Helical Structures ◽  
Force Microscopy ◽  
Ribonucleoprotein Complexes ◽  
Atomic Force ◽  
Viral Genomic Rna

AbstractThe single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.

scholarly journals Molecular architecture of the SYCP3 fibre and its interaction with DNA

Open Biology ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 190094 ◽  
Author(s):  
Daniel Bollschweiler ◽  
Laura Radu ◽  
Luay Joudeh ◽  
Jürgen M. Plitzko ◽  
Robert M. Henderson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Local Geometry ◽  
Molecular Architecture ◽  
Lateral Element ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force ◽  
Chromosome Axis

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

scholarly journals High resolution atomic force microscopy of double-stranded RNA

Nanoscale ◽  
2016 ◽  
Vol 8 (23) ◽  
pp. 11818-11826 ◽  
Author(s):  
Pablo Ares ◽  
Maria Eugenia Fuentes-Perez ◽  
Elías Herrero-Galán ◽  
José M. Valpuesta ◽  
Adriana Gil ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
High Resolution ◽  
Double Stranded Rna ◽  
Force Microscopy ◽  
Helical Pitch ◽  
Atomic Force

Imaging double-stranded RNA at sub-helical pitch resolution with atomic force microscopy in liquid.

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