The True Story and Advantages of RNA Phage Capsids as Nanotools (original) (raw)
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RNA Phage VLP-Based Vaccine Platforms
Pharmaceuticals
Virus-like particles from a variety of RNA bacteriophages have turned out to be useful platforms for delivery of vaccine antigens in a highly immunogenic format. Here we update the current state of development of RNA phage VLPs as platforms for presentation of diverse antigens by genetic, enzymatic, and chemical display methods.
Immunogenic Display of Diverse Peptides on Virus-like Particles of RNA Phage MS2
Journal of Molecular Biology, 2008
The high immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were efficiently suppressed by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLPdisplayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of 6-, 8-and 10-amino acid insertions. Not only do MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, but they also encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.
Recombinant rna phage qβ capsid particles synthesized and self-assembled in escherichia coli
Gene, 1993
The Escherichia coli RNA phage Qp coat protein-encoding gene (C) was amplified from native Qp RNA using a reverse transcription-PCR technique. Gene C contains sequences coding for both the 133-amino acid (aa) QP coat protein (CP) and the 329-aa read-through protein (Al) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon. Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared. An amplified 1062-bp PCR fragment was positioned under the control of the strong E. coli trp promoter (I',,.,) within a pGEM-derived plasmid. The synthesis of gene C products was confirmed electrophoretically and immunologically. An immunodiffusion test with anti-Q0 phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Qp C gene was responsible for high-level synthesis and correct self-assembly of QP CP monomers into capsids indistinguishable morphologically and immunologically from QP phage particles, which we plan to use as surface display vectors.
Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods
Febs Letters, 2000
Icosahedral virus-like particles (VLPs) of RNA phage QL L are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between L L-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasisix-fold symmetry contacts of the VLPs. In order to reduce the stability of QL L VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like QL L VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4^30 times the diameter of icosahedral QL L VLPs. ß
Single-stranded RNA phages ToC
Single-Stranded RNA Phages: From Molecular Biology to Nanotechnology, 2020
This item is eligible for FREE Click and Collect without a minimum order subject to availability. Details This is a comprehensive guide to single-stranded RNA phages (family Leviviridae), first discovered in 1961. These phages played a unique role in early studies of molecular biology, the genetic code, translation, replication, suppression of mutations. Special attention is devoted to modern applications of the RNA phages and their products in nanotechnology, vaccinology, gene discovery, evolutionary and environmental studies. Included is an overview of the generation of novel vaccines, gene therapy vectors, drug delivery, and diagnostic tools exploring the role of RNA phage-derived products in the revolutionary progress of the protein tethering and bioimaging protocols. Key Features Presents the first full guide to single-stranded RNA phages Reviews the history of molecular biology summarizing the role RNA phages in the development of the life sciences Demonstrates how RNA phage-derived products have resulted in nanotechnological applications Presents an up-to-date account of the role played by RNA phages in evolutionary and environmental studies
Recombinant Hepatitis E virus like particles can function as RNA nanocarriers
Journal of Nanobiotechnology, 2015
Background: Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. Findings: We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5′ region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5′-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5′-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. Conclusions: These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.
RNA nanotechnology to build a dodecahedral genome of single-stranded RNA virus
RNA Biology, 2021
The quest for artificial RNA viral complexes with authentic structure while being non-replicative is on its way for the development of viral vaccines. RNA viruses contain capsid proteins that interact with the genome during morphogenesis. The sequence and properties of the protein and genome determine the structure of the virus. For example, the Pariacoto virus ssRNA genome assembles into a dodecahedron. Virus-inspired nanotechnology has progressed remarkably due to the unique structural and functional properties of viruses, which can inspire the design of novel nanomaterials. RNA is a programmable biopolymer able to self-assemble sophisticated 3D structures with rich functionalities. RNA dodecahedrons mimicking the Pariacoto virus quasi-icosahedral genome structures were constructed from both native and 2ʹ-F modified RNA oligos. The RNA dodecahedron easily self-assembled using the stable pRNA three-way junction of bacteriophage phi29 as building blocks. The RNA dodecahedron cage was further characterized by cryo-electron microscopy and atomic force microscopy, confirming the spontaneous and homogenous formation of the RNA cage. The reported RNA dodecahedron cage will likely provide further studies on the mechanisms of interaction of the capsid protein with the viral genome while providing a template for further construction of the viral RNA scaffold to add capsid proteins for the assembly of the viral nucleocapsid as a model. Understanding the self-assembly and RNA folding of this RNA cage may offer new insights into the 3D organization of viral RNA genomes. The reported RNA cage also has the potential to be explored as a novel virus-inspired nanocarrier.
Virology, 2009
The capsids of single-stranded RNA bacteriophages show remarkable structural similarity. In an attempt to test whether the coat protein (CP) from one bacteriophage could substitute for the CP of another and form mixed particles, we reassembled capsids in vitro from a mixture of different RNA phage CP dimers together with E. coli ribosomal RNA. Surprisingly, mixing CPs from phages belonging to groups I and II led to appearance of rod-like particles along with icosahedral spherical capsids, both containing a mixture of the two CPs. Rods and mixed spherical capsids containing host RNA were also obtained in vivo in bacteria expressing simultaneously fr and GA CPs. In a co-infection of the two phages, however, only authentic fr and GA virions were formed. Coat protein mutants in the FG loop were unable to assemble into rods, suggesting that these loops are involved in the formation of the aberrant particles.
Journal of Molecular Biology, 2009
The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions. Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.
Nano Letters, 2007
Lane 1: pre-stained molecular weight marker. Lane 2: 0.5µg RLP´s. Lane 3: 10µL lipid coated colloids (5% v/v; 3µm in diameter). Lane 4: 10µL PBS of the last washing step (in a total volume of 50µL) after the fusion procedure. Lane 5: 2µL RLP coated colloids (5% v v-1). Right: protein bands assigned to rubella virus proteins and protein dimers following Oker-Blom et. al, J. Virol., 1983, 46, 964. C (Capsid protein): 33kDa; E1 (envelope protein): 58kDa (53kDa non-glycosylated); E2a and E2b (envelope proteins): 47kDa and 42kDa (30kDa non-glycosylated).