scholarly journals The Mimivirus 1.2 Mb dsDNA genome is elegantly organized into a nuclear-like weapon

2021 ◽  
Author(s):  
Chantal Abergel ◽  
Alejandro Villalta Casares ◽  
Emmanuelle Quemin ◽  
Alain Schmitt ◽  
Jean-Marie Alempic ◽  
...  
Keyword(s):  
Viral Genome ◽  
Supramolecular Organization ◽  
Host Cytoplasm ◽  
Left Handed ◽  
Helical Protein ◽  
Dsdna Viruses ◽  
Icosahedral Capsid ◽  
Protein Shell ◽  
Transcription And Replication

Abstract Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses, initially isolated in Acanthamoeba1. Little is known about the organization of the viral genome inside the membrane limited nucleoid2 and whether unpacking or other rearrangements are required prior to transcription and replication. Here we show that opening of its large icosahedral capsid in vitro leads to the release of electron dense, 30 nm diameter rod-shaped objects that appear to be expelled from the particles and unwinding. We developed a purification procedure and characterized the detailed structure at various stages of decompaction using cryo-electron microscopy single particle analyses and its composition by proteomics. This revealed that the viral genome is encased into a helical protein shell surprisingly made of the two GMC-type oxydoreductases that are also the major components of the glycosylated fibrils surrounding the capsid3. The 1.2 Mb genome is folded to follow a 5- or 6-start left-handed helix, depending on the nature of the GMC-oxydoreductase, with each helical strand lining the interior of the protein shell. Proteomic analyses of the purified genomic fibre revealed the presence of several RNA polymerase subunits as well as additional proteins involved in genome compaction that can fit into the central channel of the protein shield. Such an elegant supramolecular organization represents a remarkable evolutionary solution for packaging while protecting the viral genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. We expect that a dedicated energy-driven machinery is required for the assembly of this rod-shaped giant viral chromosome and its further compaction in the membrane limited electron dense nucleoid, characteristic of the mature Mimivirus particles2,4,5.The parsimonious implication of the same protein in two functionally unrelated substructures of the virion is also unexpected for a giant virus with a thousand genes at its disposal.

scholarly journals Structure of large dsDNA viruses

2014 ◽  
Vol 395 (7-8) ◽  
pp. 711-719 ◽  
Author(s):  
Thomas Klose ◽  
Michael G. Rossmann
Keyword(s):  
Capsid Protein ◽  
Double Layer ◽  
Viral Genome ◽  
Major Capsid Protein ◽  
Layer Membrane ◽  
Dsdna Viruses ◽  
Icosahedral Capsid ◽  
Jelly Roll ◽  
Unique Vertex ◽  
Core Genes

Abstract Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

scholarly journals In Vitro Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain

2010 ◽  
Vol 84 (18) ◽  
pp. 9350-9358 ◽  
Author(s):  
Alexis Huet ◽  
James F. Conway ◽  
Lucienne Letellier ◽  
Pascale Boulanger
Keyword(s):  
Self Assembly ◽  
Scaffolding Protein ◽  
Accessory Proteins ◽  
Conformational Diversity ◽  
In Vitro Assembly ◽  
20 Nm ◽  
Head Protein ◽  
Bacteriophage T5 ◽  
Icosahedral Capsid

ABSTRACT The Siphoviridae coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Δ domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Δ domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca2+ promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Δ domain. The T5 head maturation protease pb11 specifically cleaves the Δ domain of contracted and expanded tubes. Ca2+ is not required for proteolytic activity but for the organization of the Δ domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble in vitro without the requirement of the portal and accessory proteins. Furthermore, Ca2+ plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out in vitro, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.

Supramolecular assemblies of the Ascaris suum major sperm protein (MSP) associated with amoeboid cell motility

1994 ◽  
Vol 107 (10) ◽  
pp. 2941-2949
Author(s):  
K.L. King ◽  
M. Stewart ◽  
T.M. Roberts
Keyword(s):  
Ascaris Suum ◽  
Leading Edge ◽  
Intrinsic Property ◽  
Basic Unit ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Amoeboid Cell ◽  
Left Handed ◽  
Self Association

Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of locomotion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

scholarly journals De novo design of self-assembling helical protein filaments

Science ◽  
2018 ◽  
Vol 362 (6415) ◽  
pp. 705-709 ◽  
Author(s):  
Hao Shen ◽  
Jorge A. Fallas ◽  
Eric Lynch ◽  
William Sheffler ◽  
Bradley Parry ◽  
...  
Keyword(s):  
De Novo ◽  
Computational Design ◽  
Building Blocks ◽  
Repeat Units ◽  
Self Assembling ◽  
Wide Range ◽  
Helical Protein ◽  
Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

scholarly journals Oligonucleotides DNA containing 8-trifluoromethyl-2′-deoxyguanosine for observing Z-DNA structure

2020 ◽  
Author(s):  
Hong-Liang Bao ◽  
Tatsuki Masuzawa ◽  
Takanori Oyoshi ◽  
Yan Xu
Keyword(s):  
Genetic Diseases ◽  
Dna Structure ◽  
2D Nmr ◽  
Living Cells ◽  
Molecular Probes ◽  
Alternative Form ◽  
Left Handed ◽  
Z Dna ◽  
And Function

Abstract Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2′-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA.

scholarly journals HIV-1 uncoating occurs via a series of rapid biomechanical changes in the core related to individual stages of reverse transcription

2021 ◽  
Author(s):  
Sanela Rankovic ◽  
Akshay Deshpande ◽  
Shimon Harel ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
Morphological Changes ◽  
Viral Capsid ◽  
Target Cells ◽  
Viral Core ◽  
Successful Infection ◽  
Capsid Shell ◽  
Hiv 1

AbstractThe HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reactions containing the capsid-stabilizing host metabolite IP6. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10-30, 40-80, and 120-160 minutes after initiation of reverse transcription. Addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, thus establishing that both result from reverse transcription. Using timed addition of efavirenz, and analysis of an RNAseH-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid AFM imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. Our results suggest that reverse-transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.

scholarly journals Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

2009 ◽  
Vol 83 (10) ◽  
pp. 5137-5147 ◽  
Author(s):  
Hiromichi Hara ◽  
Hideki Aizaki ◽  
Mami Matsuda ◽  
Fumiko Shinkai-Ouchi ◽  
Yasushi Inoue ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Genome ◽  
Proteome Analysis ◽  
Virus Genome ◽  
Genome Replication ◽  
Creatine Kinase B ◽  
Efficient Replication

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

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