83 research outputs found

    The Cleavable Carboxyl-Terminus of the Small Coat Protein of Cowpea Mosaic Virus Is Involved in RNA Encapsidation

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    AbstractThe site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs

    Redox-active ferrocene-modified Cowpea mosaic virus nanoparticles

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    A naturally occurring nanoparticle, the plant virus Cowpea mosaic virus, can be decorated with ferrocene derivatives, of various linker lengths with amine and carboxylategroups, on the external surface using a range of conjugation strategies. The multiple, organometallic, redox-active ferrocene moieties on the outer surface of the virus are electrochemically independent with reduction potentials that span a potential window of 0.16 V that are dependent on the site of modification and the nature of the ferrocene derivative. The number of ferrocenes coupled to each virus ranges from about 100 to 240 depending upon the conjugation site and the linker length and these redox active units can provide multielectron reservoirs

    Combining high-resolution cryo-electron microscopy and mutagenesis to develop cowpea mosaic virus for bionanotechnology

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    Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers

    Engineering Cowpea Mosaic Virus RNA-2 into a Vector to Express Heterologous Proteins in Plants

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    AbstractA series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side of the insert, either by duplicating the MP-L cleavage site or by introducing a sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. Eight amino acids derived from the C-terminus of the MP and 14–19 amino acids from the N-terminus of the L coat protein were necessary for efficient processing of the artificial Gln/Met sites. Insertion of the FMDV 2A sequence at the C-terminus of the GFP resulted in a genetically stable construct, which produced particles containing about 10 GFP-2A-L fusion proteins. Immunocapture experiments indicated that some of the GFP is present on the virion surface. Direct fusion of GFP to the C-terminus of the S coat protein resulted in a virus which was barely viable. However, when the sequence of GFP was linked to the C-terminus by an active FMDV 2A sequence, a highly infectious construct was obtained

    Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

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    Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34 nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids

    Viruses of protozoan parasites and viral therapy: Is the time now right?

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    Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases

    Revealing the density of encoded functions in a viral RNA

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    Nikesh Patel, et al, ‘Revealing the density of encoded functions in a viral RNA’, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 112 (7): 2227-2232, February 2015, doi: http:dx.doi.org/10. 1073/pnas.1420812112. This article is freely available online through the PNAS open access option.We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogensPeer reviewedFinal Published versio

    LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection

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    The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow μLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc μL−1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection

    Crystal structure and proteomics analysis of empty virus-like particles of Cowpea mosaic virus

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    Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C-terminus of the small (S) subunit. The intact C-terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C-termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed

    Plant virus particles carrying tumour antigen activate TLR7 and induce high levels of protective antibody

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    Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-? by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant
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