Nucleoprotein Particles Research Articles

Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display.

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.

Hydrogelated Virus Nanoparticles in Tissue Engineering

Abstract: Hydrogels are an integrated three-dimensional network of water-absorbing hydrophilic polymers that can support tissue regeneration and release medication under controlled conditions. Hydrogel-based structures physically resemble the extracellular matrix besides being effective for biomedical applications and tissue engineering. Hydrogels must provide relevant biological signals to control the cell behavior to become an ideal bioactive scaffold for tissue regeneration. Incorporating virus nanoparticles (VNP) that can release essential peptides into the hydrogels is a promising option to formulate a bioactive scaffold that can facilitate cell proliferation, adhesion, migration, and differentiation. Over the recent period, virologists have discovered many viruses that lead to a great understanding of the diversity of viruses in nature. Viruses affecting the plants are called plant viruses, and they have a wide variety of shapes and sizes, yet each species produces monodisperse nucleoprotein particles. Plant viruses are not capable of infecting or reproducing in humans. Therefore, VNPs are engineered from plant viruses whose genetically programmed structures can be manipulated at the gene level, bioconjugated, or encapsulated. Plant VNPs can act as clinical diagnostic agents, immunomodulators, medicines, nanoreactors, and biosensors by displaying protein molecules or epitopes, constructing inorganic hybrid materials, or carrying molecular charges. The present review focuses on the plant virusmediated nanoparticles encapsulated in bioactive hydrogels to provide an ideal platform for tissue engineering.

Viruses and viral epidemics in the metabolic theory of evolution

Viruses, including the SARS-CoV-2 virus responsible for the current COVID-19 epidemic, are a key to the understanding of life and evolution. Cells may have arisen from aqueous sequestration inside a lipid envelope studded with chromophores capable of capturing solar photons. Nitrogen incorporation in the primordial cell chemistry allowed synthesis of amino acids and nucleic acids, a prelude to RNA and subsequently DNA. Metagenomics provides access to nucleoprotein sediments synthesised by a googol of metabolically differentiated cells that have marked the evolution of life. Replication of a virus, a nucleoprotein particle, occurs passively in competent cells. Viruses are only identified in the context of the epidemic that they induce as a result of transmission from one host to another. By breaking down the viral particle, the host cell appears to resurrect the metabolic function of the nucleic acid, which synthesises its components without any form of control. Viral products undergo self-assembly and are exported by either exocytosis or cytolysis. In the absence of cells, viruses appear to be inert. However, intracellular contamination of a virus does not always result in replication: the viral genome can disappear, remain latent, wake up, remain embedded in the cellular genome, become an oncogene or induce auto-immunity. The presence of endogenous retroviruses in eukaryotic cells raises the question of their possible role in evolution.

From infection to healing: The use of plant viruses in bioactive hydrogels.

Plant viruses show great diversity in shape and size, but each species forms unique nucleoprotein particles that are symmetrical and monodisperse. The genetically programed structure of plant viruses allows them to be modified by genetic engineering, bioconjugation, or encapsulation to form virus nanoparticles (VNPs) that are suitable for a broad range of applications. Plant VNPs can be used to present foreign proteins or epitopes, to construct inorganic hybrid materials, or to carry molecular cargos, allowing their utilization as imaging reagents, immunomodulators, therapeutics, nanoreactors, and biosensors. The medical applications of plant viruses benefit from their inability to infect and replicate in human cells. The structural properties of plant viruses also make them useful as components of hydrogels for tissue engineering. Hydrogels are three-dimensional networks composed of hydrophilic polymers that can absorb large amounts of water. They are used as supports for tissue regeneration, as reservoirs for controlled drug release, and are found in contact lenses, many wound healing materials, and hygiene products. They are also useful in ecological applications such as wastewater treatment. Hydrogel-based matrices are structurally similar to the native extracellular matrix (ECM) and provide a scaffold for the attachment of cells. To fully replicate the functions of the ECM it is necessary to augment hydrogels with biological cues that regulate cellular interactions. This can be achieved by incorporating functionalized VNPs displaying ligands that influence the mechanical characteristics of hydrogels and their biological properties, promoting the survival, proliferation, migration, and differentiation of embedded cells. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6.

BackgroundVault is the largest nonicosahedral cytosolic nucleoprotein particle, which is widely involved in induction of chemoresistance and lead to failure in long-term chemotherapy. Vault contains three different major vault proteins (MVPs) and four vault RNAs paralogues (vtRNAs, vtRNA1-1, vtRNA1-2, vtRNA1-3 and vtRNA2-1). Disruption of the MVPs do not induce hypersensitivity while expression of vtRNAs contributes to cells’ drug resistance, indicates that vtRNAs, but not MVPs play an important role in causing drug resistance. Polypyrimidine tract binding protein associated splicing factor (PSF) contributes to cell sensitivity to chemotherapy by its transcriptional activity, promotes us to figure out its potential association with vtRNAs.MethodsWe investigate the interaction between PSF and vtRNAs by electrophoretic mobility shift assays (EMSA) and RNA-immunoprecipitation (IP), and showed the binding between PSF and vtRNAs. Chromatin Immunoprecipitation (ChIP) was performed to detect the effects of vtRNAs on the interaction of PSF with GAGE6 promoter. The role of vtRNAs on chemoresistance in MCF-7 was detected by CCK-8 and EdU staining. The independent role of vtRNAs with MVP is detected by MVP or vtRNAs knockdown.ResultsThe complex with vtRNA1-1 releases PSF, allowing transcription of GAGE6 to proceed. Then we showed that induction of GAGE6 caused drug resistance by promoting cell proliferation and colony formation in soft agar. Ectopic expression of shRNA targets to vtRNA1-1 further confirmed the role of vtRNA1-1 in regulating PSF transcriptional activity independent with the expression of MVP. By vtRNA1-1 or MVP knockdown, it is revealed that vtRNA1-1 caused chemoresistance independent of MVP. Furthermore, knockdown of GAGE6 does not cause drug resistance, indicates the GAGE6 is directly involved in cell proliferation, but not the drug resistance.ConclusionThese results suggest that vtRNAs regulates cell proliferation, drug resistance, and possibly other physiological processes of humans, by complex formation with PSF.

On the structural features of influenza A nucleoprotein particles from small-angle X-ray scattering data

The structure of ribonucleic particles of influenza A virus of the A/California/07/09pdm strain is investigated by transmission electron microscopy and small-angle X-ray scattering. The small-angle X-ray scattering data obtained at room temperature correspond to previously reported data of ribonucleic particles of this virus. At higher temperatures, noticeable changes in the morphology of ribonucleic complexes are observed.

A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

Geminivirus protein structure and function

SummaryGeminiviruses are a family of plant viruses that cause economically important plant diseases worldwide. These viruses have circular single‐stranded DNA genomes and four to eight genes that are expressed from both strands of the double‐stranded DNA replicative intermediate. The transcription of these genes occurs under the control of two bidirectional promoters and one monodirectional promoter. The viral proteins function to facilitate virus replication, virus movement, the assembly of virus‐specific nucleoprotein particles, vector transmission and to counteract plant host defence responses. Recent research findings have provided new insights into the structure and function of these proteins and have identified numerous host interacting partners. Most of the viral proteins have been shown to be multifunctional, participating in multiple events during the infection cycle and have, indeed, evolved coordinated interactions with host proteins to ensure a successful infection. Here, an up‐to‐date review of viral protein structure and function is presented, and some areas requiring further research are identified.

Structural studies of large nucleoprotein particles, vaults

Vault is the largest nonicosahedral cytosolic nucleoprotein particle ever described. The widespread presence and evolutionary conservation of vaults suggest important biologic roles, although their functions have not been fully elucidated. X-ray structure of vault from rat liver was determined at 3.5 Å resolution. It exhibits an ovoid shape with a size of 40 × 40 × 67 nm3. The cage structure of vault consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The other components of vaults, telomerase-associated proteins, poly(ADP-ribose) polymerases and small RNAs, are in location in the vault particle by electron microscopy.

Evolution and Protein Packaging of Small-Molecule RNA Aptamers

A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

The chloroplasts genome (plastome) occurs at high copy numbers per cell. Several chloroplast genome copies are densely packed into nucleoprotein particles called nucleoids. How genome packaging occurs and which proteins organize chloroplast nucleoids are largely unknown. Here, we have analyzed the Chlamydomonas reinhardtii homolog of the bacterial architectural DNA-binding protein HU, the histone-like protein HLP. We show that the Chlamydomonas HLP protein is targeted to chloroplasts and associates with nucleoids. Knockdown of HLP gene expression by RNA interference (RNAi) alters the structure of chloroplast nucleoids and appears to reduce the level of compaction of chloroplast DNA. Unexpectedly, also chloroplast genome copy numbers are significantly decreased in the RNAi strains, suggesting that, in addition to its architectural role in nucleoid formation, the HLP protein is also involved in chloroplast genome maintenance.

Remodeling of the Human Papillomavirus Type 11 Replication Origin into Discrete Nucleoprotein Particles and Looped Structures by the E2 Protein

Remodeling of the Human Papillomavirus Type 11 Replication Origin into Discrete Nucleoprotein Particles and Looped Structures by the E2 Protein

Remodeling of the Human Papillomavirus Type 11 Replication Origin into Discrete Nucleoprotein Particles and Looped Structures by the E2 Protein

The human papillomavirus (HPV) DNA replication origin ( ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.

Ribosome biogenesis and the translation process in Escherichia coli.

Translation, the decoding of mRNA into protein, is the third and final element of the central dogma. The ribosome, a nucleoprotein particle, is responsible and essential for this process. The bacterial ribosome consists of three rRNA molecules and approximately 55 proteins, components that are put together in an intricate and tightly regulated way. When finally matured, the quality of the particle, as well as the amount of active ribosomes, must be checked. The focus of this review is ribosome biogenesis in Escherichia coli and its cross-talk with the ongoing protein synthesis. We discuss how the ribosomal components are produced and how their synthesis is regulated according to growth rate and the nutritional contents of the medium. We also present the many accessory factors important for the correct assembly process, the list of which has grown substantially during the last few years, even though the precise mechanisms and roles of most of the proteins are not understood.

Characterization of DNA intermediates of an arising geminivirus

Weeds of the genus Sida collected in Brazil have harbored several geminiviruses persistently over decades of vegetative propagation. They serve as cradles for new geminiviruses originating from pseudorecombination (reassortment) or molecular recombination, as has been exemplified by Sida micrantha mosaic-associated viruses (SimMV). One of such viruses has developed recently and naturally by recombination between a DNA A and a DNA B of different ancestors. We used two-dimensional gel electrophoresis and hybridization to visualize viral DNA intermediates in mixed infections as well as after transfer of single viruses into test plants. DNA intermediates which indicate multitasking in replication (rolling circle and recombination-dependent replication) were readily detected in all cases. A conspicuous increase in multimerization of circular single-stranded (ss) DNA could be attributed to the recently recombined geminivirus, suggesting poor adaptation to the host and/or inefficient gene regulation. Consequences of the accumulation of multimeric ssDNA were analyzed using nucleoprotein particle purification and electron microscopy. SimMV nucleoprotein exhibited pleomorphic structures in addition to the typical twin particles. This report provides the first analysis of DNA intermediates of an arising geminivirus.

Image analysis of bioparticles accumulation and diamagnetic alignment in high-gradient magnetic field

Magnetic properties of biological particles are measured in high-gradient magnetic separation (HGMS) analysis, revealing the concentrating process of nucleoprotein particles, ferritin, red blood cells, and eggs. A magnetic force acting on micrometer and submicrometer biological particles having diamagnetic or paramagnetic susceptibility with respect to the solution causes their movement and accumulation in gradient magnetic fields dependent on the values of the magnetic moments. The methods developed enable us to obtain the magnetic moments values of single particles and their assembly directly from magnetic separation and image analyses without assuming the detection of sizes. Our precision methods for the measurement of the capture traveling (magnetic diffusion) time and the accumulation (magnetic sedimentation) radius in HGMS show that it is really possible to determine the weak dia- or paramagnetic shifts of magnetic susceptibility up to 0.7x10(-10) (SI units). HGMS analysis of the concentrating process of nucleoprotein granules (microcells, DNA granules, or nucleosome core particles) with polarization microscopy reveals phase transitions for DNA in granules, and separation accumulation of particles enables the determination of the diamagnetic susceptibility and anisotropy properties. Magnetic concentration effects always occur in living systems because micrometer-located gradient magnetic fields inside an organism are strong enough to cause drifts of cellular complexes and organelles of micrometer and submicrometer sizes. We report the appearance of superparamagnetic contamination inside developing shrimp eggs. In the developing shrimps eggs, ferritin aggregates are observed under weak gradient magnetic fields and diaparaferromagnetic changes are detected. A significant interruption of egg development is revealed in such fields.

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts—the mating junction—between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.

Evidence for assembly-dependent folding of protein and RNA in an icosahedral virus

Ordered nucleic acid in an icosahedral virus was first visualized in the X-ray structure of the Picorna-like plant virus, Bean pod mottle virus (BPMV). Virus particles containing the 3500 nucleotide segment of the BPMV bipartite RNA genome (middle component) had nearly 20% of the genome ordered. Here we report the refined structures of the middle component, bottom component (particles containing the 5800 nucleotide segment of the genome), and top component (empty particles of BPMV capsid protein). The bottom component particles contain ordered RNA in the same location as middle component. Although the ordered RNA density in both nucleoprotein particles is the average of the contents of 60 icosahedral asymmetric units, both nucleoprotein components show that the base density for the first two nucleotides is predominantly purine, while the next five appear to be predominantly pyrimidine. The empty capsid demonstrates that RNA dictates the order of the N-terminal 19 residues of the large subunit because these residues are invisible in the top component.

Remodeling of sperm chromatin after fertilization involves nucleosomes formed by sperm histones H2A and H2B and two CS histone variants.

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.