Filamentous Virus-like Particles Research Articles

Identification of novel inhibitors against VP40 protein of Marburg virus by integrating molecular modeling and dynamics approaches

Marburg virus (MV) is a highly etiological agent of haemorrhagic fever in humans and has spread across the world. Its outbreaks caused a 23–90% human death rate. However, there are currently no authorized preventive or curative measures yet. VP40 is the MV matrix protein, which builds protein shell underneath the viral envelope and confers hallmark filamentous. VP40 alone is able to induce assembly and budding of filamentous virus-like particles (VLPs), which resemble authentic virions. As a result, this research is credited with clarifying the function of VP40 and leading to the discovery of new therapeutic targets effective in combating MV disease (MVD). Virtual screening, molecular docking and molecular dynamics (MD) simulation were used to find the putative active chemicals based on a 3D pharmacophore model of the protein’s active site cavity. Initially, andrographidine-C, a potent inhibitor was selected for the development of the pharmacophore model. Later, a library of 30,000 compounds along with the andrographidine-C was docked against VP40 protein. Three best hits including avanafil, diuvaretin and macrourone were subjected to further MD simulation analysis, as these compounds had better binding affinities as compared to andrographidine-C. Furthermore, throughout the 100 ns simulations, the back bone of VP40 protein in presence of avanafil, diuvaretin and macrourone remained stable which was further validated by MM-PBSA analysis. Additionally, all of these compounds depict maximum drug-like properties. The predicted drugs based on the ligand, avanafil, diuvaretin and macrourone could be exploited and developed as an alternative or complementary therapy for the treatment of MVD. Communicated by Ramaswamy H. Sarma

Filamentous virus-like particles are present in coral dinoflagellates across genera and ocean basins

Filamentous viruses are hypothesized to play a role in stony coral tissue loss disease (SCTLD) through infection of the endosymbiotic dinoflagellates (Family Symbiodiniaceae) of corals. To evaluate this hypothesis, it is critical to understand the global distribution of filamentous virus infections across the genetic diversity of Symbiodiniaceae hosts. Using transmission electron microscopy, we demonstrate that filamentous virus-like particles (VLPs) are present in over 60% of Symbiodiniaceae cells (genus Cladocopium) within Pacific corals (Acropora hyacinthus, Porites c.f. lobata); these VLPs are more prevalent in Symbiodiniaceae of in situ colonies experiencing heat stress. Symbiodiniaceae expelled from A. hyacinthus also contain filamentous VLPs, and these cells are more degraded than their in hospite counterparts. Similar to VLPs reported from SCTLD-affected Caribbean reefs, VLPs range from ~150 to 1500 nm in length and 16–37 nm in diameter and appear to constitute various stages in a replication cycle. Finally, we demonstrate that SCTLD-affected corals containing filamentous VLPs are dominated by diverse Symbiodiniaceae lineages from the genera Breviolum, Cladocopium, and Durusdinium. Although this study cannot definitively confirm or refute the role of filamentous VLPs in SCTLD, it demonstrates that filamentous VLPs are not solely observed in SCTLD-affected corals or reef regions, nor are they solely associated with corals dominated by members of a particular Symbiodiniaceae genus. We hypothesize that filamentous viruses are a widespread, common group that infects Symbiodiniaceae. Genomic characterization of these viruses and empirical tests of the impacts of filamentous virus infection on Symbiodiniaceae and coral colonies should be prioritized.

Characterization of a variant isolate of Zucchini yellow mosaic virus infecting green kabocha (Cucurbita maxima L.) in Bogor, Indonesia

Zucchini yellow mosaic virus (ZYMV) has been reported in Indonesia over the past decade. However, the biological, morphological, and molecular characterizations of Indonesian isolates of this virus have not yet been determined. In this study, we report a ZYMV isolate infecting green kabocha (Cucurbita maxima L.; GK) and exhibiting a typical chlorotic mosaic with vein banding symptoms. A bioassay performed on indicator plants demonstrated that the host range of the ZYMV-GK isolate was restricted to Cucurbitaceae, Amaranthaceae, and one Solanaceae family member, Nicotiana benthamiana. Interestingly, Luffa acutangula, a species in the Cucurbitaceae family, could not be infected with ZYMV-GK. Electron microscopy revealed the presence of filamentous virus-like particles, approximately 900 nm in length, characteristic to potyviruses. Sequence analysis of the coat protein gene confirmed that ZYMV-GK was a variant of ZYMV sharing 94.5% (96.7%) maximum nucleotide (amino acid) sequence identity with the GenBank sequence of a Vietnamese isolate (Acc.no. DQ925447.1).

Molecular characterization of a novel wheat-infecting virus of the family Betaflexiviridae.

Wheat plants showing yellowing and mosaic in leaves and stunting were collected from wheat fields in Henan Province, China. Analysis of these plants by transmission electron microscopy showed that they contained two types of filamentous virus-like particles with a length of 200-500 nm and 1000-1300 nm, respectively. RNA-seq revealed a coinfection with wheat yellow mosaic virus (WYMV) and an unknown wheat-infecting virus. The genome of the unknown virus is 8,410 nucleotides long, excluding its 3' poly(A) tail. It has six open reading frames (ORFs). ORF1 encodes a putative viral replication-associated protein (Rep), and ORFs 2, 3, and 4 encode the triple gene block (TGB) proteins. ORFs 5 and 6 encode the capsid protein (CP) and a protein with unknown function, respectively. Phylogenetic analysis showed that this novel virus is evolutionarily related to members of the subfamily Quinvirinae, family Betaflexiviridae. It is, however, distinct from the viruses in the currently established genera. Based on the species and genus demarcation criteria set by the International Committee on Taxonomy of Viruses (ICTV), we tentatively name this novel virus "wheat yellow stunt-associated betaflexivirus" (WYSaBV), and we propose it to be a member of a new genus in the family Betaflexiviridae.

Isolation and Characterization of Antagonistic Activity of Some Streptomycetes and their Specific Actinophages from Soil.

<b>Background and Objective:</b> Phages specific to actinomycetes are common, active in the soil and gladly detected. Soil streptomycetes are having antibiosis activities against numerous bacteria, fungi and plant viruses. Thus, this study was designed to isolate, purify and characterize some streptomycetes active against some microorganisms from soil followed by isolation of their specific phages. <b>Materials and Methods:</b> Antagonistic activities of these streptomycetes isolates were tested against <i>Bacillus subtilis</i>,<i> Pseudomonas</i> sp., <i>Serratia</i> sp. and <i>Aspergillus niger</i>. To confirm their biological characterization of the streptomycetes isolates under investigation, the 16SrRNA gene was also used. The presence of specific lysate actinophages in the soil samples were tested by spot test technique and then propagated and purified for further characterization. The morphology of the purified actinophages was determined by electron microscopy. <b>Results:</b> The five selected <i>Streptomyces</i> isolates having effective antagonistic activity were biologically and molecularly identified as <i>Streptomyces sclerogranulatus </i>(QQ06), <i>Streptomyces mutabilis </i>(QQ07), <i>Streptomyces heilongjiangensis </i>(QQ08), <i>Streptomyces sparsus </i>(QQ09) and <i>Streptomyces purpurascens </i>(QQ10) strains. Electron micrographs showed the presence of filamentous virus-like particles with lengths of 21.4×928.57, 25×750, 21.4×857.14, 21.4×885.7 and 21.4×857.14 nm specific to <i>Streptomyces</i> strains QQ06, QQ07, QQ08, QQ09 and QQ10, respectively and belong to the family Inoviridae. <b>Conclusion:</b> Phage of Inoviridae was considered as the first time against streptomycetes isolates, therefore, additional and advanced studies should be carried out at the level of molecular characterization.

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.

Engineering a Plant Viral Coat Protein for In Vitro Hybrid Self-Assembly of CO2-Capturing Catalytic Nanofilaments.

Plant virus-based nanoparticles are used as self-assembled protein scaffolds for the construction of enzyme nanocarriers. To date, one-pot production and coupling of both enzymes and scaffolds by genetic conjugation have been demonstrated only in plants. Herein, we report bacterial production and in vitro self-assembly of nanofilaments for CO2 capture. Filamentous virus-like particles (VLPs) were successfully formed by genetically fusing carbonic anhydrase from Hydrogenovibrio marinus (hmCA) to the N terminus of the coat protein (CPPVY) of potato virus Y with a flexible linker. The instability of VLPs against proteolytic degradation was circumvented by the periplasmic export of the fusion protein. The truncated form of CPPVY coexpressed by internal translation was crucial for the successful formation of long filamentous VLPs by alleviating steric hindrance via hybrid assembly. The fast and economic bottom-up fabrication of highly active nanobiocatalyst allows the nanofilaments to be efficiently used and recovered in potential biocatalytic and biosensor systems.

Entry Dynamics of Single Ebola Virus Revealed by Force Tracing

Infections by the Ebola virus (EBOV) rapidly cause fatal hemorrhagic fever in humans. Viral entry into host cells is the most critical step in infection and an attractive target for therapeutic intervention. Herein, the invagination behavior and entry dynamics of filamentous Ebola virus-like particles (EBO-VLPs) were investigated using a force tracing technique based on atomic force microscopy and single-particle fluorescence tracking in real time. The filamentous EBOV-VLPs might enter cells in both horizontal and vertical modes, and the virus-receptor interactions during endocytic uptake were analyzed. In addition, molecular dynamics simulations and engulfment energy analysis further depicted EBO-VLP entry in the horizontal and vertical directions and suggested that internalization in the vertical direction requires a larger force and more time. This report provides useful information for further revealing the mechanism of viral infection, which is important for understanding viral pathogenesis.

Comparative Study of Thermal Remodeling of Viruses with Icosahedral and Helical Symmetry

Study of the possibilities of virions and viral proteins modifications and structural remodeling is an important problem of the modern molecular virology. A technique of heat treatment of rod-shaped tobacco mosaic virus that allowed producing structurally modified spherical particles consisting of the virus coat protein was previously developed in our laboratory. These particles possessed unique adsorption and immunogenic properties and were successfully used to develop a new candidate vaccine against rubella virus. Later, the possibility of thermal remodeling of the filamentous virions of potato virus X was demonstrated. The present work reports a comparative study of thermal remodeling of viruses with different structure belonging to various taxonomic groups. The generation of structurally modified spherical particles by the heat treatment of rod-shaped virions with helical symmetry (dolichos enation mosaic virus and barley stripe mosaic virus) has been demonstrated. The dependence of the size of spherical particles derived from dolichos enation mosaic virus on the initial virus concentration was revealed. The process of thermal remodeling of the filamentous virions and virus-like particles of alternanthera mosaic virus was studied. Heat treatment of plant viruses with icosahedral symmetry was shown to cause no morphological changes.

Translocation Pathway-Dependent Assembly of Streptavidin- and Antibody-Binding Filamentous Virus-Like Particles.

Compared to well-tolerated p3 fusion, the display of fast-folding proteins fused to the minor capsid p7 and the major capsid p8, as well as in vivo biotinylation of biotin acceptor peptide (AP) fused to p7, are found to be markedly inefficient using the filamentous phage. Here, to overcome such limitations, the effect of translocation pathways, amber mutation, and phage and phagemid display systems on p7 and p8 display of antibody-binding domains are examined, while comparing the level of in vivo biotinylation of AP fused to p7 or p3. Interestingly, the in vivo biotinylation of AP occurs only in p3 fusion and the fast-folding antibody-binding scaffolds fused to p7 and p8 are best displayed via a twin-arginine translocation pathway in TG1 cells. The lower the expression level of the wild-type p8 and the smaller the size of the guest protein, the better the display of Z-domain fused to the recombinant p8. The in vivo biotinylated multifunctional filamentous virus-like particles can be vertically immobilized on streptavidin (SAV)-coated microspheres to resemble cellular microvilli-like structures, which reportedly enhance protein-protein interactions due to dramatically expanded flexible surface area.

Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry.

ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+) endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env. The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed-embedded in the membrane and present at high density-on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Influence of local environmental variables on the viral consortia associated with the coral Montipora capitata from Kaneohe Bay, Hawaii, USA

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 74:251-262 (2015) - DOI: https://doi.org/10.3354/ame01743 Influence of local environmental variables on the viral consortia associated with the coral Montipora capitata from Kaneohe Bay, Hawaii, USA Scott A. Lawrence1, Shaun P. Wilkinson1, Joanne E. Davy1, William N. S. Arlidge1, Gareth J. Williams2, William H. Wilson3,5, Greta S. Aeby4, Simon K. Davy1,* 1School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand 2Scripps Institution of Oceanography, 8602 La Jolla Shores Drive, La Jolla, California 92037, USA 3Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, PO Box 380, East Boothbay, Maine 04544, USA 4Hawaii Institute of Marine Biology, PO Box 1346, Kaneohe, Hawaii 96744, USA 5Present address: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK *Corresponding author: simon.davy@vuw.ac.nz ABSTRACT: Coral-associated viruses are a component of the coral holobiont that have received attention only relatively recently. Given the global increase in the prevalence of coral disease, and the lack of positively identified etiological agents for many diseases, these virus consortia require increased investigation. Little is known about the viruses that are naturally associated with coral reefs and how they are affected by the local environment. In the present study, a short-term analysis of viral consortia associated with the coral Montipora capitata in Kaneohe Bay, Hawaii, USA, was carried out to determine the environmental factors influencing their composition. Coral surface microlayer (CSM) and seawater samples collected at 4 sites with a range of environmental characteristics were analyzed using transmission electron microscopy (TEM), and relative abundances of virus-like particle (VLP) morphotypes were correlated with environmental measurements. Relative proportions of several CSM-associated VLP types, including phages and filamentous VLPs, were correlated with water temperature, turbidity and chlorophyll a levels. In seawater samples, turbidity and temperature showed the strongest correlation, altering the proportion of Podoviridae-like, Geminiviridae-like and putative Archaeal viruses, among others. Overall VLP consortium composition differed significantly between the CSM and seawater only at the more degraded sites, suggesting that human activity may be affecting coral reef-associated virus consortia. KEY WORDS: Corals · Virus · Environmental drivers · Turbidity · Chlorophyll Full text in pdf format PreviousNextCite this article as: Lawrence SA, Wilkinson SP, Davy JE, Arlidge WNS and others (2015) Influence of local environmental variables on the viral consortia associated with the coral Montipora capitata from Kaneohe Bay, Hawaii, USA. Aquat Microb Ecol 74:251-262. https://doi.org/10.3354/ame01743 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 74, No. 3. Online publication date: March 24, 2015 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2015 Inter-Research.

Cotranslational coat protein-mediated inhibition of potyviral RNA translation.

Potato virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection. The main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step.

Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.

Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis.

BackgoundDue to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis.MethodsHMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined.ResultsHMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly.ConclusionsThese data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0198-8) contains supplementary material, which is available to authorized users.

Ball python nidovirus: a candidate etiologic agent for severe respiratory disease in Python regius.

ABSTRACTA severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease.

Membrane binding and bending in Ebola VP40 assembly and egress.

Lipid-enveloped viruses contain a lipid bilayer coat that protects their genome and helps to facilitate entry into the host cell. Filoviruses are lipid-enveloped viruses that have up to 90% clinical fatality and include Marbug (MARV) and Ebola (EBOV). These pleomorphic filamentous viruses enter the host cell through their membrane-embedded glycoprotein and then replicate using just seven genes encoded in their negative-sense RNA genome. EBOV budding occurs from the inner leaflet of the plasma membrane (PM) and is driven by the matrix protein VP40, which is the most abundantly expressed protein of the virus. VP40 expressed in mammalian cells alone can trigger budding of filamentous virus-like particles (VLPs) that are nearly indistinguishable from authentic EBOV. VP40, such as matrix proteins from other viruses, has been shown to bind anionic lipid membranes. However, how VP40 selectively interacts with the inner leaflet of the PM and assembles into a filamentous lipid enveloped particle is mostly unknown. This article describes what is known regarding VP40 membrane interactions and what answers will fill the gaps.

인삼(Panax ginseng)에 발생한 Watermelon mosaic virus의 새로운 병원성 계통

A disease, supposedly caused by a virus, was observed from Insam (Panax ginseng) fields of Punggi in year 2006. It has long believed to be a physiological disorder. However, the incidence of the disease has increased every year. When several samples were observed under electron microscope, filamentous virus-like particles were observed. The nucleotide sequences of the virus were analyzed by RT-PCR with specific primer sets derived from the results of DNA chip. The results indicated that the disease was caused by Watermelon mosaic virus (WMV). It revealed that the result of the biological assay by the virus was different from that of WMV previously found in other crops. Therefore, this is the first report that WMV causes the disease in P. ginseng and the virus is named to be WMV-Insam.

Precision Templating with DNA of a Virus-like Particle with Peptide Nanostructures

We report here the preparation of filamentous virus-like particles by the encapsulation of a linear or circular double-stranded DNA template with preassembled mushroom-shaped nanostructures having a positively charged domain. These nanostructures mimic the capsid proteins of natural filamentous viruses and are formed by self-assembly of coiled-coil peptides conjugated at opposite termini with cationic segments and poly(ethylene glycol) (PEG) chains. We found that a high molecular weight of PEG segments was critical for the formation of monodisperse and uniformly shaped filamentous complexes. It is proposed that electrostatic attachment of the nanostructures with sufficiently long PEG segments generates steric forces that increase the rigidity of the neutralized DNA template. This stiffening counterbalances the natural tendency of the DNA template to condense into toroids or buckle multiple times. The control achieved over both shape and dimensions of the particles offers a strategy to create one-dimensional supramolecular nanostructures of defined length containing nucleic acids.