Filamentous Virus Research Articles

Molecular characterization of the garlic virus B genome and evidence of allexivirus recombination

Molecular characterization is important for differentiating allexiviruses species, since detection by serological methods may be uncertain. Eight different species have been reported as infecting garlic: Garlic virus A, B, C, D, E, X (GarV-A, B, C, D, E, X), Shallot virus X (ShVX) and Garlic mite-borne filamentous virus (GarMbFV), and the complete genome is known for six of these. This work reports for the first time the complete sequence of GarV-B and makes a phylogenetic and recombination analysis between the different allexivirus species. Total RNA was obtained of a GarV-B positive garlic plant by ISEM-D using anti-GarV-B antiserum and this was sent for mass sequencing. Deep sequencing revealed the first complete GarV-B genome, consisting of 8327 nucleotides (nt). The genome contained six open reading frames (ORFs) with the typical genome organization which encodes putative proteins of 168 kDa (ORF1), 27 kDa (ORF2), 12 kDa (ORF3), 39 kDa (ORF4), 27 kDa (ORF5) and 14 kDa (ORF6). The comparison of the gene coding for the coat protein of the virus showed a greater identity of nt with other isolates of GarV-B (88.4 to 99.7%) and of GarV-X (75.4 to 78.3%) published in GenBank. The GarV-B replicase gene has not been previously reported in GenBank, so the sequence was compared with GarV-A, -C, −D, −E, −X and ShVX. The highest nt identity values were detected with isolates of GarV-X (73.5 to 74.1%) and GarV-C (71.9 to 72.8%). These results suggest that GarV-X and GarV-B may be different strains of the same virus. A genetic recombination analysis was also performed between the complete sequences of allexiviruses published and obtained in this work and it was detected that the species GarV-D and GarV-E may have arisen from the recombination of the N-terminal portion of GarV-B with the C-terminal portion of GarV-A.

Controllable Assembly of Flexible Protein Nanotubes for Loading Multifunctional Modules.

Viruses with filamentous morphologies, such as tobacco mosaic virus (TMV) and M13 bacteriophage, have long been studied as multivalent nanoscaffolds for loading functional motifs. Structural assembly of the capsid proteins (CPs) of filamentous viruses often requires the presence of DNA or RNA molecules, which has limited their applications. Here, we describe a strategy for controllable assembly of flexible bio-nanotubes consisting of Escherichia coli expressed CP of baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV) in vitro. These protein-only nanotubes were studied as a new structural platform for high-density presentation of multiple active molecules on the exterior surface by direct fusion of the protein of interest to the N-terminus of HearNPV CP (HaCP). Structural characterization using cryoelectron microscopy demonstrated that the HaCP could assemble into two closely related but structurally distinct tube types, suggesting the tunable HaCP interaction network is the major contributor to the flexibility of HaCP nanotubes. Our flexible nanotubes could tolerate larger molecular modifications compared with TMV-based templates and could be used as promising candidates for versatile molecular loading applications.

Amino-acids-functionalized M13-assisted synthesis of silver and silver sulfide nanoparticles

Bio-mineralization has been explored to synthesize nanomaterials with exquisite structures and amazing properties. Amino-acids-functionalized filamentous M13 virus was applied as a scaffold for the synthesis of silver and silver sulfide nanoparticles. It is interesting to find that the binding affinity between filamentous M13 virus and silver ions can be effectively improved after a friendly modification by amino acids. At the same time, amino-acids-modified filamentous M13 virus also serves as a dispersant to decrease the size and increase the stability of silver nanoparticles. Both silver and silver sulfide nanoparticles with monodispersed size and uniform morphology can be fabricated by the direction of amino-acid-modified filamentous M13 virus. The successful synthesis of silver sulfide nanoparticles on amino-acids-modified filamentous M13 virus suggests that this approach is highly efficient to fabricate not only metal nanoparticles, but also binary or multinary compounds.

Detection of Israeli Acute Paralysis Virus (IAPV) and Apis mellifera Filamentous Virus (AmFV) in Honey Bees in Mexico

Abstract The recent alarming loss of honey bee colonies around the world is believed to be related to the presence of viruses. The aim of this study was to detect two major viral diseases, Apis mellifera Filamentous virus (AmFV) and Israeli Acute Paralysis Virus (IAPV) using Reverse Transcription - Polymerase Chain Reaction RT-PCR, in honey bees in Mexico. Adult and larvae honey bee samples were collected from asymptomatic colonies of six major beekeeping regions in the state of Chihuahua, Mexico. Both viruses were detected in both developmental stages of honey bees, IAPV at a higher prevalence (23.5%) as compared to AmFV, only in 0.9% of samples. However, this is the first report on AmFV infection in Mexican apiaries. Further studies are required to understand the AmFV and IAPV impact on colony loss in Mexico and to develop strategies for enhancing the control of viral diseases.

Deciphering the behaviour manipulation imposed by a virus on its parasitoid host: insights from a dual transcriptomic approach.

Behaviour manipulation imposed by parasites is a fascinating phenomenon but our understanding is still very limited. We studied the interaction between a virus and the parasitic wasp Leptopilina boulardi that attacks Drosophila larvae. Wasps usually refrain to lay eggs into already parasitized hosts (superparasitism avoidance). On the contrary, females infected by the Leptopilina boulardi Filamentous Virus (LbFV) are much more incline to superparasitize. Interestingly, the host-sharing induced by this behaviour modification leads to the horizontal transmission of the virus, thus increasing its fitness at the expense of that of the wasp. To better understand the mechanisms underlying this behaviour manipulation, we studied by RNA sequencing the meta-transcriptome of LbFV and the parasitic wasp both in the abdomen and in the head. We found that the abundance of viral transcripts was independent of the wasp strain but strongly differed between tissues. Based on the tissue pattern of expression, we identified a set of 20 viral genes putatively involved in the manipulation process. In addition, we identified a set of wasp genes deregulated in the presence of the virus either in the abdomen or in the head, including genes with annotations suggesting involvement in behaviour (i.e. Potassium-channel protein). This dataset gives new insights into the behaviour manipulation and on the genetic basis of superparasitism in parasitoids.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Dynamics and Rigidity of an Intact Filamentous Bacteriophage Virus Probed by Magic Angle Spinning NMR

The capsid dynamics of filamentous bacteriophages is related to their function, stability, and interactions with the genome, and can be assessed by measuring the chemical shift anisotropy (CSA) of 15 N amides, which are sensitive to large amplitude motions. In this study, CSA recoupling experiments under magic-angle spinning NMR were used to probe the dynamics of the y21m capsid mutant of fd bacteriophage. Based on fitting the generated CSA lineshapes, residues located in the N-terminus undergo increased motional amplitudes suggesting its global motion, whereas other backbone residues are rigid, and imply a tight hydrophobic packing of the phage.

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.

The Virus Bioresistor: Wiring Virus Particles for the Direct, Label-Free Detection of Target Proteins.

The virus bioresistor (VBR) is a chemiresistor that directly transfers information from virus particles to an electrical circuit. Specifically, the VBR enables the label-free detection of a target protein that is recognized and bound by filamentous M13 virus particles, each with dimensions of 6 nm ( w) × 1 μm ( l), entrained in an ultrathin (∼250 nm) composite virus-polymer resistor. Signal produced by the specific binding of virus to target molecules is monitored using the electrical impedance of the VBR: The VBR presents a complex impedance that is modeled by an equivalent circuit containing just three circuit elements: a solution resistance ( Rsoln), a channel resistance ( RVBR), and an interfacial capacitance ( CVBR). The value of RVBR, measured across 5 orders of magnitude in frequency, is increased by the specific recognition and binding of a target protein to the virus particles in the resistor, producing a signal Δ RVBR. The VBR concept is demonstrated using a model system in which human serum albumin (HSA, 66 kDa) is detected in a phosphate buffer solution. The VBR cleanly discriminates between a change in the electrical resistance of the buffer, measured by Rsoln, and selective binding of HSA to virus particles, measured by RVBR. The Δ RVBR induced by HSA binding is as high as 200 Ω, contributing to low sensor-to-sensor coefficients-of-variation (<15%) across the entire calibration curve for HSA from 7.5 nM to 900 nM. The response time for the VBR is 3-30 s.

Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study

Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane.

Antigen-Antibody Interaction-Based Self-Healing Capability of Hybrid Hydrogels Composed of Genetically Engineered Filamentous Viruses and Gold Nanoparticles.

Filamentous M13 phages have recently been utilized as components for developing novel functional soft materials in various fields such as sensor, device, and biomedical applications. Recently, we have developed liquid crystalline hydrogels composed of M13 phages and gold nanoparticles (GNPs) based on specific interactions between the components. The main objective of this study was to clarify the self-healing capability of the hydrogels composed of M13 phages and GNPs. M13 phages displaying tag peptides with a sequence of YPYDVPDYA (HA phages) were genetically constructed through general molecular biology. The mechanical strength of hydrogels composed of the HA phages and anti-HA peptide antibodies-immobilized GNPs (HA-GNPs) was measured by indentation tests. The rupture point of the hydrogels was visually observed. An aliquot of buffer solution was added into the rupture point of the hydrogels after the indentation test. After incubation for 2 days, self-healing of the rupture point was checked visually. The indentation test was also performed after self-healing. To clarify the assembled structures of the components in the hydrogels, transmission electron microscopy (TEM) observation was performed by transferring the hydrogel onto a TEM grid before and after healing. The strength of the original hydrogel (before self-healing) required for rupture was approximately 55 mN. Self-healing of the rupture point was confirmed visually, and the hydrogels behaved as uniform hydrogels again during the vial inversion tests. As a result of the indentation test for the self-healed points of the hydrogels, the rupture force of approximately 45 mN was detected, indicating the self-healing capability of the hydrogels. TEM observation of the before and after self-healing exhibited the regularly assembled structures composed of the HA-GNPs, suggesting that the ruptured networks were recovered into regularly assembled network structures. Importantly, control of the concentration of the HA-GNPs resulted in suppression of decreasing the rupture forces during the repetitive self-healing processes. Our results demonstrated the self-healing capability of structurally regular hybrid hydrogels composed of genetically engineered filamentous viruses displaying antigen peptides and antibody-immobilized GNPs. The results indicated that supramolecular hydrogels containing filamentous viruses would expand the applicability of virus-based soft materials.

Filamentous Virus-based Assembly: Their Oriented Structures and Thermal Diffusivity

Organic polymers are generally regarded as thermal insulators because amorphous arrangement of molecular chains reduces the mean free path of heat-conducting phonons. However, recent studies indicated that single chains of polymers with highly oriented structures could have high thermal conductivity than bulk polymers because stretched polymer chains effectively conduct phonons through polymeric covalent bonds. Here, we demonstrated the possibility of non-covalent virus assembly prepared by simple flow-induced methods toward high thermal conductive polymeric materials. Films with high thermal diffusivity composed of non-covalent bond-based assemblies of liquid crystalline filamentous viruses were prepared using a simple flow-induced orientation method. Structural and thermal characterization demonstrated that highly oriented structures of the viruses in the film were attributed to the high thermal diffusivity. Our results will open attractive opportunities for biomolecular-based thermally conductive soft materials even though the assemblies are based on non-covalent bonds.

Confinement-induced liquid crystalline transitions in amyloid fibril cholesteric tactoids.

Chirality is ubiquitous in nature and plays crucial roles in biology, medicine, physics and materials science. Understanding and controlling chirality is therefore an important research challenge with broad implications. Unlike other chiral colloids, such as nanocellulose or filamentous viruses, amyloid fibrils form nematic phases but appear to miss their twisted form, the cholesteric or chiral nematic phases, despite a well-defined chirality at the single fibril level. Here we report the discovery of cholesteric phases in amyloids, using β-lactoglobulin fibrils shortened by shear stresses. The physical behaviour of these new cholesteric materials exhibits unprecedented structural complexity, with confinement-driven ordering transitions between at least three types of nematic and cholesteric tactoids. We use energy functional theory to rationalize these results and observe a chirality inversion from the left-handed amyloids to right-handed cholesteric droplets. These findings deepen our understanding of cholesteric phases, advancing their use in soft nanotechnology, nanomaterial templating and self-assembly.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys224, 225 and Lys274, 275). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes.

Buckling Causes Nonlinear Dynamics of Filamentous Viruses Driven through Nanopores.

Measurements and Langevin dynamics simulations of filamentous viruses driven through solid-state nanopores reveal a superlinear rise in the translocation velocity with driving force. The mobility also scales with the length of the virus in a nontrivial way that depends on the force. These dynamics are consequences of the buckling of the leading portion of a virus as it emerges from the nanopore and is put under compressive stress by the viscous forces it encounters. The leading tip of a buckled virus stalls and this reduces the total viscous drag force. We present a scaling theory that connects the solid mechanics to the nonlinear dynamics of polyelectrolytes translocating nanopores.

The study of toxicity and pathogenicity risk of Potato Virus X/Herceptin nanoparticles as agents for cancer therapy

BackgroundNowadays, viral nanoparticles (VNPs) have attracted a lot of attention, although some are concerned that VNPs may reflect on viral entry of virus into animal or plant cells. In the present study, it aimed to investigate the pathogenesis of VNPs formed by Herceptin (Trastuzumab) and the viral nanoparticles derived from the filamentous plant virus Potato Virus X (PVX-HER) in host plant.MethodsFlow cytometry was used to evaluate the toxicity of plant virus alone (free-PVX) in two HER2+ cancer cells and normal cells. Furthermore, the pathogenesis of PVX-HER was tested by ELISA, Western blot, and RT-PCR.ResultsThe results indicated that PVX-HER failed to cause disease in the host plant. Furthermore, cancer and normal cell lines could evade the apoptosis and necrosis by the free viral nanoparticles (free-PVX). This study suggests that the problem of toxicity considered as a challenging factor in most nanoparticles was not observed in this plant virus nanoparticle.ConclusionsFinally, the safety of the environment was confirmed. The present research is the first study which focused on VNPs based on safety perspective that can be used for drug delivery carriers.

Filamentous Viruses as Building Blocks for Hierarchical Self-Assembly toward Functional Soft Materials

Abstract This review describes the utilization of M13 phage, one of the filamentous viruses, for the development of novel functional soft materials. Traditionally, M13 phage has been widely used as a scaffold to display peptides or proteins on the surface of M13 phage through genetic engineering. This technology is well known as “phage display”, and generally used nowadays for the construction of peptide or protein libraries on M13 phage surfaces to identify peptides or proteins with desired functions. Recently, construction of hierarchical assembled structures composed of M13 phage as a building block has generated great interest as a means of using the excellent properties of M13 phage for the development of novel classes of soft materials. In this case, liquid crystalline formation of M13 phage is effectively used to fabricate structurally regulated assemblies. Further combination of the phage display technique and liquid crystalline formation widely expands the applicability of M13 phage to various fields such as devices, sensors, and biomedicines. Such approaches to M13 phage should contribute to novel attractive opportunities for next-generation soft materials for science and technology.

In Planta Production of Fluorescent Filamentous Plant Virus-Based Nanoparticles.

Viral nanoparticles are attractive platforms for biomedical applications and are frequently employed for optical imaging in tissue culture and preclinical animal models as fluorescent probes. Chemical modification with organic dyes remains the most common strategy to develop such fluorescent probes. Here we report a genetic engineering approach to incorporate fluorescent proteins in viral nanoparticles, which can be propagated in their plant host. The fluorescent viral nanoparticles so obtained obviate post-harvest modifications and thereby maximize yields. Our engineering approach transforms filamentous potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions at a 1:3 fusion protein to CP ratio through integration of the foot-and-mouth disease 2A sequence. The in planta propagation of recombinant GFP-PVX or mCherry-PVX thus produced in Nicotiana benthamiana can be easily documented using fluorescence imaging. Molecular farming protocols can be accordingly optimized by monitoring chimera stability over the course of the infection cycle. Moreover, we also demonstrate the utility of recombinant mCherry-PVX in optical imaging of human cancer cells and tumor tissue in preclinical mice model. Together, these features make genetically engineered fluorescent PVX particles ideally suited for molecular imaging applications.

Filamentous Bacteriophage Proteins and Assembly.

Filamentous bacteriophages, also known as filamentous bacterial viruses or Inoviruses, have been studied extensively over the years. They are interesting paradigms in structural molecular biology and offer insight into molecular assembly, a process that remains to be fully understood. In this chapter, an overview on filamentous bacteriophages will be provided. In particular, we review the constituent proteins of filamentous bacteriophage and discuss assembly by examining the structure of the major coat protein at various stages of the process. The minor coat proteins will also be briefly reviewed. Structural information provides key snapshots into the dynamic process of assembly.

Structural Homology Between Nucleoproteins of ssRNA Viruses.

Our understanding of the viral world changed just after the first structures of icosahedral viral particles were unveiled. The structural similarities between capsid proteins of distant viral groups were not anticipated, and the findings suggested the existence of common ancestors for viruses with different host range, genomic structure and multiplication strategies. This way, diverse viruses with icosahedral particles can now be grouped based on the structural homology between their capsid proteins. In the last years, the presence of conserved folds between viral proteins in non-icosahedral viruses has also emerged. Viral particles with radically different morphologies, ranging from naked and filamentous to enveloped and pleomorphic, have shown structural homology between the nucleoproteins that bind directly to their genomes. This chapter overviews recent findings regarding the similar structure found between nucleoproteins of eukaryotic ssRNA viruses. The structural homology includes the coat proteins from all known families of flexible filamentous plant viruses, a group with monopartite (+)ssRNA genomes. Their coat proteins share a core domain with nucleoproteins of previously unrelated families of enveloped viruses that have segmented (-)ssRNA genomes. This last group consists of mostly animals viruses, including influenza virus.